Tertiary Amine Coupling by Oxidation for Selective Labeling of Dimethyl Lysine Post-Translational Modifications.

Lysine dimethylation (Kme2) is a crucial post-translational modification (PTM) that regulates biological processes and is implicated in diseases. There is significant interest in globally identifying these methylation marks. Unfortunately, this remains challenging due to the lack of robust technologies for selectively labeling Kme2. To address this, we present a chemical method named tertiary amine coupling by oxidation (TACO). This method selectively modifies Kme2 to aldehydes using Selectfluor and a base. The resulting aldehydes from Kme2 were then functionalized using reductive amination, thiolamine, and oxime chemistry. We successfully demonstrated the versatility of TACO in selectively labeling Kme2 peptides and proteins in complex cell lysate mixtures with varying payloads, including affinity tags and fluorophores. We further showed the application of TACO chemistry for the identification of Kme2 sites at a single-molecule level by fluorosequencing. We discovered novel 30 Kme2 sites, in addition to previously known 5 Kme2 sites, by proteomics analysis of TACO-modified nuclear extracts. Our work establishes a unique strategy for covalently modifying Kme2, facilitating the global identification of low-abundance Kme2-PTMs and their sites within complex cell lysate mixtures.

Targeting TRIP13 in favorable histology Wilms tumor with nuclear export inhibitors synergizes with doxorubicin.

Wilms tumor (WT) is the most common renal malignancy of childhood. Despite improvements in the overall survival, relapse occurs in ~15% of patients with favorable histology WT (FHWT). Half of these patients will succumb to their disease. Identifying novel targeted therapies remains challenging in part due to the lack of faithful preclinical in vitro models. Here we establish twelve patient-derived WT cell lines and demonstrate that these models faithfully recapitulate WT biology using genomic and transcriptomic techniques. We then perform loss-of-function screens to identify the nuclear export gene, XPO1, as a vulnerability. We find that the FDA approved XPO1 inhibitor, KPT-330, suppresses TRIP13 expression, which is required for survival. We further identify synergy between KPT-330 and doxorubicin, a chemotherapy used in high-risk FHWT. Taken together, we identify XPO1 inhibition with KPT-330 as a potential therapeutic option to treat FHWTs and in combination with doxorubicin, leads to durable remissions in vivo.

Tunable fluorescent probes for detecting aldehydes in living systems.

Aldehydes, pervasive in various environments, pose health risks at elevated levels due to their collective toxic effects via shared mechanisms. Monitoring total aldehyde content in living systems is crucial due to their cumulative impact. Current methods for detecting cellular aldehydes are limited to UV and visible ranges, restricting their analysis in living systems. This study introduces an innovative reaction-based trigger that leverages the exceptional selectivity of 2-aminothiophenol for aldehydes, leading to the production of dihydrobenzothiazole and activating a fluorescence response. Using this trigger, we developed a series of fluorescent probes for aldehydes by altering the fluorophore allowing for excitation and emission wavelengths across the visible to near-infrared spectral regions without compromising the reactivity of the bioorthogonal moiety. These probes exhibit remarkable aldehyde chemoselectivity, rapid kinetics, and high quantum yields, enabling the detection of diverse aldehyde types, both exogenous and endogenous, within complex biological contexts. Notably, we employed the most red-shifted near-infrared probe from this series to detect aldehydes in living systems, including biliary organoids and mouse organs. These probes provide valuable tools for exploring the multifaceted roles of aldehydes in biological functions and diseases within living systems, laying the groundwork for further investigations.

Exploring the Molecular Underpinnings of Cancer-Causing Oncohistone Mutants Using Yeast as a Model.

Understanding the molecular basis of cancer initiation and progression is critical in developing effective treatment strategies. Recently, mutations in genes encoding histone proteins that drive oncogenesis have been identified, converting these essential proteins into "oncohistones". Understanding how oncohistone mutants, which are commonly single missense mutations, subvert the normal function of histones to drive oncogenesis requires defining the functional consequences of such changes. Histones genes are present in multiple copies in the human genome with 15 genes encoding histone H3 isoforms, the histone for which the majority of oncohistone variants have been analyzed thus far. With so many wildtype histone proteins being expressed simultaneously within the oncohistone, it can be difficult to decipher the precise mechanistic consequences of the mutant protein. In contrast to humans, budding and fission yeast contain only two or three histone H3 genes, respectively. Furthermore, yeast histones share ~90% sequence identity with human H3 protein. Its genetic simplicity and evolutionary conservation make yeast an excellent model for characterizing oncohistones. The power of genetic approaches can also be exploited in yeast models to define cellular signaling pathways that could serve as actionable therapeutic targets. In this review, we focus on the value of yeast models to serve as a discovery tool that can provide mechanistic insights and inform subsequent translational studies in humans.

Chemical sensors for imaging total cellular aliphatic aldehydes in live cells.

Aliphatic aldehydes are reactive electrophilic carbonyls that cross-link with DNA and proteins leading to cellular toxicity and disease pathogenesis. This toxicity is due to the cooperative effect of multiple aldehydes via a common mechanism. Therefore, live-cell imaging of total aliphatic aldehydes, small-to-long chain (C1-C10), is highly desired to decipher their physiological and pathological functions. However, sensors for imaging total cellular aliphatic aldehydes are currently lacking despite their high concentrations (∼80 to >500 µM) inside cells. Herein, we report chemical sensors that generate a benzimidazole moiety upon reaction with aliphatic aldehydes of different chain lengths (C1-C10), resulting in turn-on fluorescence. These sensors exhibit high quantum yields, high dynamic range, and enable the quantification of changes in both the exogenous administration of aldehydes and endogenous real-time formation of aliphatic aldehydes in live mammalian cells. This tool has great potential to transform aldehyde research by illuminating cellular metabolites that have remained elusive in living systems.

Emerging Role of Epigenetic Modifiers in Breast Cancer Pathogenesis and Therapeutic Response.

Breast cancer pathogenesis, treatment, and patient outcomes are shaped by tumor-intrinsic genomic alterations that divide breast tumors into molecular subtypes. These molecular subtypes often dictate viable therapeutic interventions and, ultimately, patient outcomes. However, heterogeneity in therapeutic response may be a result of underlying epigenetic features that may further stratify breast cancer patient outcomes. In this review, we examine non-genetic mechanisms that drive functional changes to chromatin in breast cancer to contribute to cell and tumor fitness and highlight how epigenetic activity may inform the therapeutic response. We conclude by providing perspectives on the future of therapeutic targeting of epigenetic enzymes, an approach that holds untapped potential to improve breast cancer patient outcomes.

Identification of ligand-receptor pairs that drive human astrocyte development.

Extrinsic signaling between diverse cell types is crucial for nervous system development. Ligand binding is a key driver of developmental processes. Nevertheless, it remains a significant challenge to disentangle which and how extrinsic signals act cooperatively to affect changes in recipient cells. In the developing human brain, cortical progenitors transition from neurogenesis to gliogenesis in a stereotyped sequence that is in part influenced by extrinsic ligands. Here we used published transcriptomic data to identify and functionally test five ligand-receptor pairs that synergistically drive human astrogenesis. We validate the synergistic contributions of TGFß2, NLGN1, TSLP, DKK1 and BMP4 ligands on astrocyte development in both hCOs and primary fetal tissue. We confirm that the cooperative capabilities of these five ligands are greater than their individual capacities. Additionally, we discovered that their combinatorial effects converge in part on the mTORC1 signaling pathway, resulting in transcriptomic and morphological features of astrocyte development. Our data-driven framework can leverage single-cell and bulk genomic data to generate and test functional hypotheses surrounding cell-cell communication regulating neurodevelopmental processes.

Adipocyte-mediated epigenomic instability in human T-ALL cells is cytotoxic and phenocopied by epigenetic-modifying drugs.

The world's population with obesity is reaching pandemic levels. If current trends continue, it is predicted that there will be 1.5 billion people with obesity by 2030. This projection is alarming due to the association of obesity with numerous diseases including cancer, with recent studies demonstrating a positive association with acute myeloid leukemia (AML) and B cell acute lymphoblastic leukemia (B-ALL). Interestingly, several epidemiological studies suggest the converse relationship may exist in patients with T cell acute lymphoblastic leukemia (T-ALL). To determine the relationship between obesity and T-ALL development, we employed the diet-induced obesity (DIO) murine model and cultured human T-ALL cells in adipocyte-conditioned media (ACM), bone marrow stromal cell-conditioned media, stromal conditioned media (SCM), and unconditioned media to determine the functional impact of increased adiposity on leukemia progression. Whereas only 20% of lean mice transplanted with T-ALL cells survived longer than 3 months post-inoculation, 50%-80% of obese mice with leukemia survived over this same period. Furthermore, culturing human T-ALL cells in ACM resulted in increased histone H3 acetylation (K9/K14/K18/K23/K27) and methylation (K4me3 and K27me3) posttranslational modifications (PTMs), which preceded accelerated cell cycle progression, DNA damage, and cell death. Adipocyte-mediated epigenetic changes in human T-ALL cells were recapitulated with the H3K27 demethylase inhibitor GSK-J4 and the pan-HDAC inhibitor vorinostat. These drugs were also highly cytotoxic to human T-ALL cells at low micromolar concentrations. In summary, our data support epidemiological studies demonstrating that adiposity suppresses T-ALL pathogenesis. We present data demonstrating that T-ALL cell death in adipose-rich microenvironments is induced by epigenetic modifications, which are not tolerated by leukemia cells. Similarly, GSK-J4 and vorinostat treatment induced epigenomic instability and cytotoxicity profiles that phenocopied the responses of human T-ALL cells to ACM, which provides additional support for the use of epigenetic modifying drugs as a treatment option for T-ALL.

Medulloblastoma and the DNA Damage Response.

Medulloblastoma (MB) is the most common malignant brain tumor in children with standard of care consisting of surgery, radiation, and chemotherapy. Recent molecular profiling led to the identification of four molecularly distinct MB subgroups - Wingless (WNT), Sonic Hedgehog (SHH), Group 3, and Group 4. Despite genomic MB characterization and subsequent tumor stratification, clinical treatment paradigms are still largely driven by histology, degree of surgical resection, and presence or absence of metastasis rather than molecular profile. Patients usually undergo resection of their tumor followed by craniospinal radiation (CSI) and a 6 month to one-year multi-agent chemotherapeutic regimen. While there is clearly a need for development of targeted agents specific to the molecular alterations of each patient, targeting proteins responsible for DNA damage repair could have a broader impact regardless of molecular subgrouping. DNA damage response (DDR) protein inhibitors have recently emerged as targeted agents with potent activity as monotherapy or in combination in different cancers. Here we discuss the molecular underpinnings of genomic instability in MB and potential avenues for exploitation through DNA damage response inhibition.

A Saccharomyces cerevisiae model and screen to define the functional consequences of oncogenic histone missense mutations.

Somatic missense mutations in histone genes turn these essential proteins into oncohistones, which can drive oncogenesis. Understanding how missense mutations alter histone function is challenging in mammals as mutations occur in a single histone gene. For example, described oncohistone mutations predominantly occur in the histone H3.3 gene, despite the human genome encoding 15 H3 genes. To understand how oncogenic histone missense mutations alter histone function, we leveraged the budding yeast model, which contains only 2 H3 genes, to explore the functional consequences of oncohistones H3K36M, H3G34W, H3G34L, H3G34R, and H3G34V. Analysis of cells that express each of these variants as the sole copy of H3 reveals that H3K36 mutants show different drug sensitivities compared to H3G34 mutants. This finding suggests that changes to proximal amino acids in the H3 N-terminal tail alter distinct biological pathways. We exploited the caffeine-sensitive growth of H3K36-mutant cells to perform a high copy suppressor screen. This screen identified genes linked to histone function and transcriptional regulation, including Esa1, a histone H4/H2A acetyltransferase; Tos4, a forkhead-associated domain-containing gene expression regulator; Pho92, an N6-methyladenosine RNA-binding protein; and Sgv1/Bur1, a cyclin-dependent kinase. We show that the Esa1 lysine acetyltransferase activity is critical for suppression of the caffeine-sensitive growth of H3K36R-mutant cells while the previously characterized binding interactions of Tos4 and Pho92 are not required for suppression. This screen identifies pathways that could be altered by oncohistone mutations and highlights the value of yeast genetics to identify pathways altered by such mutations.

Tunable Amine-Reactive Electrophiles for Selective Profiling of Lysine.

Proteome profiling by activated esters identified >9000 ligandable lysines but they are limited as covalent inhibitors due to poor hydrolytic stability. Here we report our efforts to design and discover a new series of tunable amine-reactive electrophiles (TAREs) for selective and robust labeling of lysine. The major challenges in developing selective probes for lysine are the high nucleophilicity of cysteines and poor hydrolytic stability. Our work circumvents these challenges by a unique design of the TAREs that form stable adducts with lysine and on reaction with cysteine generate another reactive electrophiles for lysine. We highlight that TAREs exhibit substantially high hydrolytic stability as compared to the activated esters and are non-cytotoxic thus have the potential to act as covalent ligands. We applied these alternative TAREs for the intracellular labeling of proteins in different cell lines, and for the selective identification of lysines in the human proteome on a global scale.

Targeting MLL Methyltransferases Enhances the Antitumor Effects of PI3K Inhibition in Hormone Receptor-positive Breast Cancer.

The high frequency of aberrant PI3K pathway activation in hormone receptor-positive (HR+) breast cancer has led to the development, clinical testing, and approval of the p110α-selective PI3K inhibitor alpelisib. The limited clinical efficacy of alpelisib and other PI3K inhibitors is partially attributed to the functional antagonism between PI3K and estrogen receptor (ER) signaling, which is mitigated via combined PI3K inhibition and endocrine therapy. We and others have previously demonstrated chromatin-associated mechanisms by which PI3K supports cancer development and antagonizes ER signaling through the modulation of the H3K4 methylation axis, inhibition of KDM5A promoter H3K4 demethylation and KMT2D/MLL4-directed enhancer H3K4 methylation. Here we show that inhibition of the H3K4 histone methyltransferase MLL1 in combination with PI3K inhibition impairs HR+ breast cancer clonogenicity and cell proliferation. While combined PI3K/MLL1 inhibition reduces PI3K/AKT signaling and H3K4 methylation, MLL1 inhibition increases PI3K/AKT signaling through the dysregulation of gene expression associated with AKT activation. These data reveal a feedback loop between MLL1 and AKT whereby MLL1 inhibition reactivates AKT. We show that combined PI3K and MLL1 inhibition synergizes to cause cell death in in vitro and in vivo models of HR+ breast cancer, which is enhanced by the additional genetic ablation of the H3K4 methyltransferase and AKT target KMT2D/MLL4. Together, our data provide evidence of a feedback mechanism connecting histone methylation with AKT and may support the preclinical development and testing of pan-MLL inhibitors. Significance: Here the authors leverage PI3K/AKT-driven chromatin modification to identify histone methyltransferases as a therapeutic target. Dual PI3K and MLL inhibition synergize to reduce clonogenicity and cell proliferation, and promote in vivo tumor regression. These findings suggest patients with PIK3CA-mutant, HR+ breast cancer may derive clinical benefit from combined PI3K/MLL inhibition.

Practical advice for mentoring and supporting faculty colleagues in STEM fields: Views from mentor and mentee perspectives.

In 2020, the American Society of Biochemistry and Molecular Biology (ASBMB) Women in Biochemistry and Molecular Biology Committee introduced the ASBMB Leadership Awards to recognize individuals with a strong commitment to advancing the careers of women in biochemistry and molecular biology along with demonstrated excellence in research, discovery, and/or service. This innovative award recognizes efforts to mentor and support trainees and colleagues at all levels. Such a leadership award provides the opportunity to focus briefly on the important role of mentoring within the STEM disciplines. The goal of this commentary, which brings together perspectives from a senior scientist and recent recipient of the ASBMB Mid-Career Leadership Award as well as two junior faculty, is to highlight approaches for purposeful support of colleagues, with an emphasis on going beyond formal mentoring committees. The commentary primarily focuses on mentoring within the academic arena of extramural funding and publication, highlighting the reality that multiple mentors with diverse expertise and perspectives are critical to support success within STEM careers.

PIK3CA C-terminal frameshift mutations are novel oncogenic events that sensitize tumors to PI3K-α inhibition.

PIK3CA hotspot mutation is well established as an oncogenic driver event in cancer and its durable and efficacious inhibition is a focus in the development and testing of clinical cancer therapeutics. However, hundreds of cancer-associated PIK3CA mutations remain uncharacterized, their sensitivity to PI3K inhibitors unknown. Here, we describe a series of PIK3CA C-terminal mutations, primarily nucleotide insertions, that produce a frame-shifted protein product with an extended C terminus. We report that these mutations occur at a low frequency across multiple cancer subtypes, including breast, and are sufficient to drive oncogenic transformation in vitro and in vivo. We demonstrate that the oncogenicity of these mutant p110α proteins is dependent on p85 but not Ras association. P110α-selective pharmacologic inhibition blocks transformation in cells and mammary tumors characterized by PIK3CA C-terminal mutation. Taken together, these results suggest patients with breast and other tumors characterized by PIK3CA C-terminal frameshift mutations may derive benefit from p110α-selective inhibitors, including the recently FDA-approved alpelisib.

PI3K-p110α mediates the oncogenic activity induced by loss of the novel tumor suppressor PI3K-p85α.

Mutation or loss of the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K) is emerging as a transforming factor in cancer, but the mechanism of transformation has been controversial. Here we find that hemizygous deletion of the PIK3R1 gene encoding p85α is a frequent event in breast cancer, with PIK3R1 expression significantly reduced in breast tumors. PIK3R1 knockdown transforms human mammary epithelial cells, and genetic ablation of Pik3r1 accelerates a mouse model of HER2/neu-driven breast cancer. We demonstrate that partial loss of p85α increases the amount of p110α-p85 heterodimers bound to active receptors, augmenting PI3K signaling and oncogenic transformation. Pan-PI3K and p110α-selective pharmacological inhibition effectively blocks transformation driven by partial p85α loss both in vitro and in vivo. Together, our data suggest that p85α plays a tumor-suppressive role in transformation, and suggest that p110α-selective therapeutics may be effective in the treatment of breast cancer patients with PIK3R1 loss.

Epigenetic regulation of RTK signaling.

Receptor tyrosine kinase (RTK) signaling cascades coordinate intracellular signaling in response to growth factors, chemokines, and other extracellular stimuli to control fundamental biological processes such as cellular proliferation, metabolism, and survival. Hyperactivation of pathways associated with growth factor signaling (e.g., RTK and downstream effectors including Ras, PI3K/AKT, and Raf) is a frequent event in human cancers, which uncouples ligand-mediated activation with signal transduction. While the contributions of direct genomic events are well understood as causative agents of hyperactive signal transduction, other non-heritable genomic modifications promote the activation of growth factor-associated signaling cascades. In this review, we highlight epigenomic mechanisms by which hyperactivation of RTK-associated signaling cascades occurs and may contribute to cancer.

The emerging role of PI3K/AKT-mediated epigenetic regulation in cancer.

The PI3-kinase/AKT pathway integrates signals from external cellular stimuli to regulate essential cellular functions, and is frequently aberrantly activated in human cancers. Recent research demonstrates that tight regulation of the epigenome is critical in preserving and restricting transcriptional activation, which can impact cellular growth and proliferation. In this review we examine mechanisms by which the PI3K/AKT pathway regulates the epigenome to promote oncogenesis, and highlight how connections between PI3K/AKT and the epigenome may impact the future therapeutic treatment of cancers featuring a hyperactivated PI3K/AKT pathway.

PI3K/AKT Signaling Regulates H3K4 Methylation in Breast Cancer.

Post-translational histone H3 modifications regulate transcriptional competence. The mechanisms by which the epigenome is regulated in response to oncogenic signaling remain unclear. Here we show that H3K4me3 is increased in breast tumors driven by an activated PIK3CA allele and that inhibition of PI3K/AKT signaling reduces promoter-associated H3K4me3 in human breast cancer cells. We show that the H3K4 demethylase KDM5A is an AKT target and that phosphorylation of KDM5A regulates its nuclear localization and promoter occupancy. Supporting a role for KDM5A in mediating PI3K/AKT transcriptional effects, the decreased expression in response to AKT inhibition of a subset of cell-cycle genes associated with poor clinical outcome is blunted by KDM5A silencing. Our data identify a mechanism by which PI3K/AKT signaling modulates the cancer epigenome through controlling H3K4 methylation and suggest that KDM5A subcellular localization and genome occupancy may be pharmacodynamic markers of the activity of PI3K/AKT inhibitors currently in clinical development.

The HPV16 E6 oncoprotein causes prolonged receptor protein tyrosine kinase signaling and enhances internalization of phosphorylated receptor species.

The high-risk human papillomavirus (HPV) E6 proteins are consistently expressed in HPV-associated lesions and cancers. HPV16 E6 sustains the activity of the mTORC1 and mTORC2 signaling cascades under conditions of growth factor deprivation. Here we report that HPV16 E6 activated mTORC1 by enhanced signaling through receptor protein tyrosine kinases, including epidermal growth factor receptor and insulin receptor and insulin-like growth factor receptors. This is evidenced by sustained signaling through these receptors for several hours after growth factor withdrawal. HPV16 E6 increased the internalization of activated receptor species, and the signaling adaptor protein GRB2 was shown to be critical for HPV16 E6 mediated enhanced EGFR internalization and mTORC1 activation. As a consequence of receptor protein kinase mediated mTORC1 activation, HPV16 E6 expression increased cellular migration of primary human epithelial cells. This study identifies a previously unappreciated mechanism by which HPV E6 proteins perturb host-signaling pathways presumably to sustain protein synthesis during the viral life cycle that may also contribute to cellular transforming activities of high-risk HPV E6 proteins.

The human papillomavirus type 8 E6 protein interferes with NOTCH activation during keratinocyte differentiation.

Cutaneous ß-human papillomavirus (ß-HPV) E6 proteins inhibit NOTCH signaling by associating with the transcriptional coactivator MAML1. NOTCH has tumor suppressor activities in epithelial cells and is activated during keratinocyte differentiation. Here we report that HPV type 8 (HPV8) E6 subverts NOTCH activation during keratinocyte differentiation by inhibiting RBPJ/MAML1 transcriptional activator complexes at NOTCH target DNA. NOTCH inhibition impairs epithelial differentiation and may thus contribute to ß-HPV replication and viral oncogenesis.