Secreted Wnt proteins regulate development and adult tissue homeostasis by binding and activating cell-surface Frizzled receptors and co-receptors including LRP5/6. The hydrophobicity of Wnt proteins has complicated their purification and limited their use in basic research and as therapeutics. We describe modular tetravalent antibodies that can recruit Frizzled and LRP5/6 in a manner that phenocopies the activities of Wnts both in vitro and in vivo. The modular nature of these synthetic Frizzled and LRP5/6 Agonists, called FLAgs, enables tailored engineering of specificity for one, two or multiple members of the Frizzled family. We show that FLAgs underlie differentiation of pluripotent stem cells, sustain organoid growth, and activate stem cells in vivo. Activation of Wnt signaling circuits with tailored FLAgs will enable precise delineation of functional outcomes directed by distinct receptor combinations and could provide a new class of therapeutics to unlock the promise of regenerative medicine.

The positive transcription elongation factor (P-TEFb) (CDK9/cyclin T1) regulates RNA Polymerase II dependent transcription of cellular and integrated viral genes. It is an essential cofactor for HIV-1 Tat-transactivation and selective inhibition of P-TEFb blocks HIV-1 replication without affecting cellular transcription, indicating that P-TEFb could be a potential target for developing anti-HIV therapeutics. Flavopiridol, a small molecule CDK inhibitor, blocks HIV-1 Tat-transactivation and viral replication by inhibiting P-TEFb kinase activity, but it is highly cytotoxic. In search for selective and less cytotoxic P-TEFb inhibitors, we prepared a series of flavopiridol analogues and evaluated their kinase inhibitory activity against P-TEFb and CDK2/cyclin A, cellular antiviral potency, and cytotoxicity. We identified several analogues that selectively inhibit P-TEFb kinase activity in vitro and show antiviral potency comparable to that of flavopiridol, but with significantly reduced cytotoxicity. These compounds are valuable molecular probes for understanding P-TEFb-regulated cellular and HIV-1 gene transcription and provide potential anti-HIV-1 therapeutics.

Activation of Wnt signaling entails βcatenin protein stabilization and translocation to the nucleus to regulate context-specific transcriptional programs. The majority of colorectal cancers (CRCs) initiate following APC mutations, resulting in Wnt ligand—independent stabilization and nuclear accumulation of βcatenin. The mechanisms underlying βcatenin nucleocytoplasmic shuttling remain incompletely defined. Using a novel, positive selection, functional genomic strategy, DEADPOOL, we performed a genome-wide CRISPR screen and identified IPO11 as a required factor for βcatenin-mediated transcription in APC mutant CRC cells. IPO11 (Importin-11) is a nuclear import protein that shuttles cargo from the cytoplasm to the nucleus. IPO11−/− cells exhibit reduced nuclear βcatenin protein levels and decreased βcatenin target gene activation, suggesting IPO11 facilitates βcatenin nuclear import. IPO11 knockout decreased colony formation of CRC cell lines and decreased proliferation of patient-derived CRC organoids. Our findings uncover a novel nuclear import mechanism for βcatenin in cells with high Wnt activity.

Transcription of HIV-1 genes depends on the RNA polymerase II kinase and elongation factor positive transcription elongation factor b (P-TEFb), the complex of cyclin T1 and CDK9. Recent evidence suggests that regulation of transcription by P-TEFb involves chromatin binding and modifying factors. To determine how P-TEFb may connect chromatin remodeling to transcription, we investigated the relationship between P-TEFb and histone H1. We identify histone H1 as a substrate for P-TEFb involved in cellular and HIV-1 transcription. We show that P-TEFb interacts with H1 and that P-TEFb inhibition by RNAi, flavopiridol, or dominant negative CDK9 expression correlates with loss of phosphorylation and mobility of H1 in vivo. Importantly, P-TEFb directs H1 phosphorylation in response to wild-type HIV-1 infection, but not Tat-mutant HIV-1 infection. Our results show that P-TEFb phosphorylates histone H1 at a specific C-terminal phosphorylation site. Expression of a mutant H1.1 that cannot be phosphorylated by P-TEFb also disrupts Tat transactivation in an HIV reporter cell line as well as transcription of the c-fos and hsp70 genes in HeLa cells. We identify histone H1 as a novel P-TEFb substrate, and our results suggest new roles for P-TEFb in both cellular and HIV-1 transcription.The transcription of many viral and eukaryotic genes is controlled at the point of mRNA elongation by positive transcription elongation factor b (P-TEFb), 2 the complex of CDK9 and cyclin T1. This P-TEFb control has been shown in the HIV-1 genes encoding proviral peptides (1, 2), oncogenes such as c-fos (3), and inducible genes such as hsp70 (4). The transcription of these genes is initiated by RNA polymerase II (Pol II), but is inhibited shortly thereafter by Pol II pausing (1, 2). This pause is alleviated by P-TEFb, which increases Pol II processivity by hyperphosphorylating the Ser-2 sites of its C-terminal domain, thus promoting the transition from abortive to productive mRNA elongation (1,2). Importantly, HIV-1 transcription can be prevented by inhibiting P-TEFb phosphorylation of Pol II, such as by siRNA-mediated knockdown of CDK9 or by treating infected cells with the potent CDK9 inhibitor, flavopiridol (5, 6).Active P-TEFb also binds to multiple chromatin-binding and -modifying proteins such as the bromodomain-containing protein Brd4. Brd4 recruits P-TEFb to promoters by binding both acetylated histone proteins and components of the Mediator complex (7-9). P-TEFb not only interacts with Brd4 at cellular promoters, but also binds specific transcription factors such as NFB during cellular and HIV transcription (10, 11). Thus, P-TEFb may function in coupling chromatin modification to transcription.Histone H1 is a highly abundant linker histone protein that binds to nucleosomes and the DNA that connects them (12). H1 facilitates the compaction of chromatin, thus maintaining chromatin patterns during differentiation and development (13,14). In addition to its role in chromatin compaction, H1 exists in equilibrium between chromatin-bound and -fre...

Positive transcription elongation factor b (P-TEFb), the complex of Cyclin T1 and CDK9, activates the transcription of many viral and eukaryotic genes at the point of mRNA elongation. The activity of P-TEFb has been implicated in the differentiation of a number of cell types, including skeletal muscle. In order to promote transcription, P-TEFb hyperphosphorylates RNA Pol II, thereby increasing its processivity. Our previous work identified histone H1 as a P-TEFb substrate during HIV-1 and immediate-early transcription. Here, we examine the role of P-TEFb phosphorylation of histone H1 during differentiation, using the myoblast cell line C2C12 as a model for skeletal muscle differentiation. We found that H1 phosphorylation is elevated in differentiating C2C12, and this phosphorylation is sensitive to P-TEFb inhibition. H1 phosphorylation was also necessary for the induction of three muscle marker genes that require P-TEFb for expression. Additionally, ChIP experiments demonstrate that H1 dissociates from muscle differentiation marker genes in C2C12 cells under active P-TEFb conditions. We determine that both P-TEFb activity and H1 phosphorylation are necessary for the full differentiation of C2C12 myoblasts into myotubes.

FBXW7, which encodes a substrate-specific receptor of an SCF E3 ligase complex, is a frequently mutated human tumor suppressor gene known to regulate the post-translational stability of various proteins involved in cellular proliferation. Here, using genomewide CRISPR screens, we report a novel synthetic lethal genetic interaction between FBXW7 and CCNL1 and describe CCNL1 as a new substrate of the SCF-FBXW7 E3 ligase. Further analysis showed that the CCNL1-CDK11 complex is critical at the G2-M phase of the cell cycle since defective CCNL1 accumulation, resulting from FBXW7 mutation, leads to shorter mitotic time. Cells harboring FBXW7 loss-of-function mutations are hypersensitive to treatment with a CDK11 inhibitor, highlighting a genetic vulnerability that could be leveraged for cancer treatment.

Intratumoral heterogeneity helps drive the selection for diverse therapy-resistant cell populations. In this study, we demonstrate the coexistence of two therapy-resistant populations with distinct properties that are reproducibly enriched under conditions that characterize tumor pathophysiology. Breast cancer cells that survived chemotherapy or hypoxia were enriched for cells expressing the major hyaluronic acid receptor CD44. However, only CD44 hi cells that survived chemotherapy exhibited cancer stem cell (CSC) phenotypes based on growth potential and gene expression signatures that represent oncogenic signaling and metastatic prowess. Strikingly, we identified TGFb2 as a key growth promoter of CD44 hi cells that survived chemotherapy but also as a growth inhibitor of cells that survived hypoxia. Expression of the TGFb receptor TGFbR1 and its effector molecule SMAD4 was required for enrichment of CD44 hi cells exposed to the chemotherapeutic drug epirubicin, which suggests a feed-forward loop to enrich for and enhance the function of surviving CSCs. Our results reveal context-dependent effects of TGFb2 signaling in the same tumor at the same time. The emergence of distinct resistant tumor cell populations as a consequence of prior therapeutic intervention or microenvironmental cues has significant implications for the responsiveness of recurring tumors to therapy.

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