A Benzarone Derivative Inhibits EYA to Suppress Tumor Growth in SHH Medulloblastoma.

Medulloblastoma is one of the most common malignant brain tumors of children, and 30% of medulloblastomas are driven by gain-of-function genetic lesions in the Sonic Hedgehog (SHH) signaling pathway. EYA1, a haloacid dehalogenase phosphatase and transcription factor, is critical for tumorigenesis and proliferation of SHH medulloblastoma (SHH-MB). Benzarone and benzbromarone have been identified as allosteric inhibitors of EYA proteins. Using benzarone as a point of departure, we developed a panel of 35 derivatives and tested them in SHH-MB. Among these compounds, DS-1-38 functioned as an EYA antagonist and opposed SHH signaling. DS-1-38 inhibited SHH-MB growth in vitro and in vivo, showed excellent brain penetrance, and increased the lifespan of genetically engineered mice predisposed to fatal SHH-MB. These data suggest that EYA inhibitors represent promising therapies for pediatric SHH-MB. SIGNIFICANCE: Development of a benzarone derivative that inhibits EYA1 and impedes the growth of SHH medulloblastoma provides an avenue for improving treatment of this malignant pediatric brain cancer.

Targeting transcription factors through an IMiD independent zinc finger domain.

Immunomodulatory imide drugs (IMiDs) degrade specific C2H2 zinc finger degrons in transcription factors, making them effective against certain cancers. SALL4, a cancer driver, contains seven C2H2 zinc fingers in four clusters, including an IMiD degron in zinc finger cluster two (ZFC2). Surprisingly, IMiDs do not inhibit growth of SALL4 expressing cancer cells. To overcome this limit, we focused on a non-IMiD degron, SALL4 zinc finger cluster four (ZFC4). By combining AlphaFold and the ZFC4-DNA crystal structure, we identified a potential ZFC4 drug pocket. Utilizing an in silico docking algorithm and cell viability assays, we screened chemical libraries and discovered SH6, which selectively targets SALL4-expressing cancer cells. Mechanistic studies revealed that SH6 degrades SALL4 protein through the CUL4A/CRBN pathway, while deletion of ZFC4 abolished this activity. Moreover, SH6 led to significant 62% tumor growth inhibition of SALL4+ xenografts in vivo and demonstrated good bioavailability in pharmacokinetic studies. In summary, these studies represent a new approach for IMiD independent drug discovery targeting C2H2 transcription factors in cancer.

Irisin acts through its integrin receptor in a two-step process involving extracellular Hsp90α.

Exercise benefits the human body in many ways. Irisin is secreted by muscle, increased with exercise, and conveys physiological benefits, including improved cognition and resistance to neurodegeneration. Irisin acts via αV integrins; however, a mechanistic understanding of how small polypeptides like irisin can signal through integrins is poorly understood. Using mass spectrometry and cryo-EM, we demonstrate that the extracellular heat shock protein 90α (eHsp90α) is secreted by muscle with exercise and activates integrin αVß5. This allows for high-affinity irisin binding and signaling through an Hsp90α/αV/ß5 complex. By including hydrogen/deuterium exchange data, we generate and experimentally validate a 2.98 Å RMSD irisin/αVß5 complex docking model. Irisin binds very tightly to an alternative interface on αVß5 distinct from that used by known ligands. These data elucidate a non-canonical mechanism by which a small polypeptide hormone like irisin can function through an integrin receptor.

Carbohydrate fatty acid monosulphate: oil-in-water adjuvant enhances SARS-CoV-2 RBD nanoparticle-induced immunogenicity and protection in mice.

Development of SARS-CoV-2 vaccines that protect vulnerable populations is a public health priority. Here, we took a systematic and iterative approach by testing several adjuvants and SARS-CoV-2 antigens to identify a combination that elicits antibodies and protection in young and aged mice. While demonstrating superior immunogenicity to soluble receptor-binding domain (RBD), RBD displayed as a protein nanoparticle (RBD-NP) generated limited antibody responses. Comparison of multiple adjuvants including AddaVax, AddaS03, and AS01B in young and aged mice demonstrated that an oil-in-water emulsion containing carbohydrate fatty acid monosulphate derivative (CMS:O/W) most effectively enhanced RBD-NP-induced cross-neutralizing antibodies and protection across age groups. CMS:O/W enhanced antigen retention in the draining lymph node, induced injection site, and lymph node cytokines, with CMS inducing MyD88-dependent Th1 cytokine polarization. Furthermore, CMS and O/W synergistically induced chemokine production from human PBMCs. Overall, CMS:O/W adjuvant may enhance immunogenicity and protection of vulnerable populations against SARS-CoV-2 and other infectious pathogens.

Depletion of creatine phosphagen energetics with a covalent creatine kinase inhibitor.

Creatine kinases (CKs) provide local ATP production in periods of elevated energetic demand, such as during rapid anabolism and growth. Thus, creatine energetics has emerged as a major metabolic liability in many rapidly proliferating cancers. Whether CKs can be targeted therapeutically is unknown because no potent or selective CK inhibitors have been developed. Here we leverage an active site cysteine present in all CK isoforms to develop a selective covalent inhibitor of creatine phosphagen energetics, CKi. Using deep chemoproteomics, we discover that CKi selectively engages the active site cysteine of CKs in cells. A co-crystal structure of CKi with creatine kinase B indicates active site inhibition that prevents bidirectional phosphotransfer. In cells, CKi and its analogs rapidly and selectively deplete creatine phosphate, and drive toxicity selectively in CK-dependent acute myeloid leukemia. Finally, we use CKi to uncover an essential role for CKs in the regulation of proinflammatory cytokine production in macrophages.

High Efficacy and Drug Synergy of HDAC6-Selective Inhibitor NN-429 in Natural Killer (NK)/T-Cell Lymphoma.

NK/T-cell lymphoma (NKTCL) and γδ T-cell non-Hodgkin lymphomas (γδ T-NHL) are highly aggressive lymphomas that lack rationally designed therapies and rely on repurposed chemotherapeutics from other hematological cancers. Histone deacetylases (HDACs) have been targeted in a range of malignancies, including T-cell lymphomas. This study represents exploratory findings of HDAC6 inhibition in NKTCL and γδ T-NHL through a second-generation inhibitor NN-429. With nanomolar in vitro HDAC6 potency and high in vitro and in cellulo selectivity for HDAC6, NN-429 also exhibited long residence time and improved pharmacokinetic properties in contrast to older generation inhibitors. Following unique selective cytotoxicity towards γδ T-NHL and NKTCL, NN-429 demonstrated a synergistic relationship with the clinical agent etoposide and potential synergies with doxorubicin, cytarabine, and SNS-032 in these disease models, opening an avenue for combination treatment strategies.

BAF Complex Maintains Glioma Stem Cells in Pediatric H3K27M Glioma.

Diffuse midline gliomas are uniformly fatal pediatric central nervous system cancers that are refractory to standard-of-care therapeutic modalities. The primary genetic drivers are a set of recurrent amino acid substitutions in genes encoding histone H3 (H3K27M), which are currently undruggable. These H3K27M oncohistones perturb normal chromatin architecture, resulting in an aberrant epigenetic landscape. To interrogate for epigenetic dependencies, we performed a CRISPR screen and show that patient-derived H3K27M-glioma neurospheres are dependent on core components of the mammalian BAF (SWI/SNF) chromatin remodeling complex. The BAF complex maintains glioma stem cells in a cycling, oligodendrocyte precursor cell-like state, in which genetic perturbation of the BAF catalytic subunit SMARCA4 (BRG1), as well as pharmacologic suppression, opposes proliferation, promotes progression of differentiation along the astrocytic lineage, and improves overall survival of patient-derived xenograft models. In summary, we demonstrate that therapeutic inhibition of the BAF complex has translational potential for children with H3K27M gliomas. SIGNIFICANCE: Epigenetic dysregulation is at the core of H3K27M-glioma tumorigenesis. Here, we identify the BRG1-BAF complex as a critical regulator of enhancer and transcription factor landscapes, which maintain H3K27M glioma in their progenitor state, precluding glial differentiation, and establish pharmacologic targeting of the BAF complex as a novel treatment strategy for pediatric H3K27M glioma. See related commentary by Beytagh and Weiss, p. 2730. See related article by Mo et al., p. 2906.

Covalent disruptor of YAP-TEAD association suppresses defective Hippo signaling.

The transcription factor TEAD, together with its coactivator YAP/TAZ, is a key transcriptional modulator of the Hippo pathway. Activation of TEAD transcription by YAP has been implicated in a number of malignancies, and this complex represents a promising target for drug discovery. However, both YAP and its extensive binding interfaces to TEAD have been difficult to address using small molecules, mainly due to a lack of druggable pockets. TEAD is post-translationally modified by palmitoylation that targets a conserved cysteine at a central pocket, which provides an opportunity to develop cysteine-directed covalent small molecules for TEAD inhibition. Here, we employed covalent fragment screening approach followed by structure-based design to develop an irreversible TEAD inhibitor MYF-03-69. Using a range of in vitro and cell-based assays we demonstrated that through a covalent binding with TEAD palmitate pocket, MYF-03-69 disrupts YAP-TEAD association, suppresses TEAD transcriptional activity and inhibits cell growth of Hippo signaling defective malignant pleural mesothelioma (MPM). Further, a cell viability screening with a panel of 903 cancer cell lines indicated a high correlation between TEAD-YAP dependency and the sensitivity to MYF-03-69. Transcription profiling identified the upregulation of proapoptotic BMF gene in cancer cells that are sensitive to TEAD inhibition. Further optimization of MYF-03-69 led to an in vivo compatible compound MYF-03-176, which shows strong antitumor efficacy in MPM mouse xenograft model via oral administration. Taken together, we disclosed a story of the development of covalent TEAD inhibitors and its high therapeutic potential for clinic treatment for the cancers that are driven by TEAD-YAP alteration.

Structural basis for defective membrane targeting of mutant enzyme in human VLCAD deficiency.

Very long-chain acyl-CoA dehydrogenase (VLCAD) is an inner mitochondrial membrane enzyme that catalyzes the first and rate-limiting step of long-chain fatty acid oxidation. Point mutations in human VLCAD can produce an inborn error of metabolism called VLCAD deficiency that can lead to severe pathophysiologic consequences, including cardiomyopathy, hypoglycemia, and rhabdomyolysis. Discrete mutations in a structurally-uncharacterized C-terminal domain region of VLCAD cause enzymatic deficiency by an incompletely defined mechanism. Here, we conducted a structure-function study, incorporating X-ray crystallography, hydrogen-deuterium exchange mass spectrometry, computational modeling, and biochemical analyses, to characterize a specific membrane interaction defect of full-length, human VLCAD bearing the clinically-observed mutations, A450P or L462P. By disrupting a predicted α-helical hairpin, these mutations either partially or completely impair direct interaction with the membrane itself. Thus, our data support a structural basis for VLCAD deficiency in patients with discrete mutations in an α-helical membrane-binding motif, resulting in pathologic enzyme mislocalization.

A novel ß-catenin/BCL9 complex inhibitor blocks oncogenic Wnt signaling and disrupts cholesterol homeostasis in colorectal cancer.

Dysregulated Wnt/ß-catenin signaling is implicated in the pathogenesis of many human cancers, including colorectal cancer (CRC), making it an attractive clinical target. With the aim of inhibiting oncogenic Wnt activity, we developed a high-throughput screening AlphaScreen assay to identify selective small-molecule inhibitors of the interaction between ß-catenin and its coactivator BCL9. We identified a compound that consistently bound to ß-catenin and specifically inhibited in vivo native ß-catenin/BCL9 complex formation in CRC cell lines. This compound inhibited Wnt activity, down-regulated expression of the Wnt/ß-catenin signature in gene expression studies, disrupted cholesterol homeostasis, and significantly reduced the proliferation of CRC cell lines and tumor growth in a xenograft mouse model of CRC. This study has therefore identified a specific small-molecule inhibitor of oncogenic Wnt signaling, which may have value as a probe for functional studies and has important implications for the development of novel therapies in patients with CRC.

An adjuvant strategy enabled by modulation of the physical properties of microbial ligands expands antigen immunogenicity.

Activation of the innate immune system via pattern recognition receptors (PRRs) is key to generate lasting adaptive immunity. PRRs detect unique chemical patterns associated with invading microorganisms, but whether and how the physical properties of PRR ligands influence the development of the immune response remains unknown. Through the study of fungal mannans, we show that the physical form of PRR ligands dictates the immune response. Soluble mannans are immunosilent in the periphery but elicit a potent pro-inflammatory response in the draining lymph node (dLN). By modulating the physical form of mannans, we developed a formulation that targets both the periphery and the dLN. When combined with viral glycoprotein antigens, this mannan formulation broadens epitope recognition, elicits potent antigen-specific neutralizing antibodies, and confers protection against viral infections of the lung. Thus, the physical properties of microbial ligands determine the outcome of the immune response and can be harnessed for vaccine development.

An aluminum hydroxide:CpG adjuvant enhances protection elicited by a SARS-CoV-2 receptor binding domain vaccine in aged mice.

Global deployment of vaccines that can provide protection across several age groups is still urgently needed to end the COVID-19 pandemic, especially in low- and middle-income countries. Although vaccines against SARS-CoV-2 based on mRNA and adenoviral vector technologies have been rapidly developed, additional practical and scalable SARS-CoV-2 vaccines are required to meet global demand. Protein subunit vaccines formulated with appropriate adjuvants represent an approach to address this urgent need. The receptor binding domain (RBD) is a key target of SARS-CoV-2 neutralizing antibodies but is poorly immunogenic. We therefore compared pattern recognition receptor (PRR) agonists alone or formulated with aluminum hydroxide (AH) and benchmarked them against AS01B and AS03-like emulsion-based adjuvants for their potential to enhance RBD immunogenicity in young and aged mice. We found that an AH and CpG adjuvant formulation (AH:CpG) produced an 80-fold increase in anti-RBD neutralizing antibody titers in both age groups relative to AH alone and protected aged mice from the SARS-CoV-2 challenge. The AH:CpG-adjuvanted RBD vaccine elicited neutralizing antibodies against both wild-type SARS-CoV-2 and the B.1.351 (beta) variant at serum concentrations comparable to those induced by the licensed Pfizer-BioNTech BNT162b2 mRNA vaccine. AH:CpG induced similar cytokine and chemokine gene enrichment patterns in the draining lymph nodes of both young adult and aged mice and enhanced cytokine and chemokine production in human mononuclear cells of younger and older adults. These data support further development of AH:CpG-adjuvanted RBD as an affordable vaccine that may be effective across multiple age groups.

Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo.

The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1's active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target.

Alum:CpG adjuvant enables SARS-CoV-2 RBD-induced protection in aged mice and synergistic activation of human elder type 1 immunity.

Global deployment of vaccines that can provide protection across several age groups is still urgently needed to end the COVID-19 pandemic especially for low- and middle-income countries. While vaccines against SARS-CoV-2 based on mRNA and adenoviral-vector technologies have been rapidly developed, additional practical and scalable SARS-CoV-2 vaccines are needed to meet global demand. In this context, protein subunit vaccines formulated with appropriate adjuvants represent a promising approach to address this urgent need. Receptor-binding domain (RBD) is a key target of neutralizing antibodies (Abs) but is poorly immunogenic. We therefore compared pattern recognition receptor (PRR) agonists, including those activating STING, TLR3, TLR4 and TLR9, alone or formulated with aluminum hydroxide (AH), and benchmarked them to AS01B and AS03-like emulsion-based adjuvants for their potential to enhance RBD immunogenicity in young and aged mice. We found that the AH and CpG adjuvant formulation (AH:CpG) demonstrated the highest enhancement of anti-RBD neutralizing Ab titers in both age groups (∼80-fold over AH), and protected aged mice from the SARS-CoV-2 challenge. Notably, AH:CpG-adjuvanted RBD vaccine elicited neutralizing Abs against both wild-type SARS-CoV-2 and B.1.351 variant at serum concentrations comparable to those induced by the authorized mRNA BNT162b2 vaccine. AH:CpG induced similar cytokine and chemokine gene enrichment patterns in the draining lymph nodes of both young adult and aged mice and synergistically enhanced cytokine and chemokine production in human young adult and elderly mononuclear cells. These data support further development of AH:CpG-adjuvanted RBD as an affordable vaccine that may be effective across multiple age groups. ONE SENTENCE SUMMARY: Alum and CpG enhance SARS-CoV-2 RBD protective immunity, variant neutralization in aged mice and Th1-polarizing cytokine production by human elder leukocytes.

Binding and transport of SFPQ-RNA granules by KIF5A/KLC1 motors promotes axon survival.

Complex neural circuitry requires stable connections formed by lengthy axons. To maintain these functional circuits, fast transport delivers RNAs to distal axons where they undergo local translation. However, the mechanism that enables long-distance transport of RNA granules is not yet understood. Here, we demonstrate that a complex containing RNA and the RNA-binding protein (RBP) SFPQ interacts selectively with a tetrameric kinesin containing the adaptor KLC1 and the motor KIF5A. We show that the binding of SFPQ to the KIF5A/KLC1 motor complex is required for axon survival and is impacted by KIF5A mutations that cause Charcot-Marie Tooth (CMT) disease. Moreover, therapeutic approaches that bypass the need for local translation of SFPQ-bound proteins prevent axon degeneration in CMT models. Collectively, these observations indicate that KIF5A-mediated SFPQ-RNA granule transport may be a key function disrupted in KIF5A-linked neurologic diseases and that replacing axonally translated proteins serves as a therapeutic approach to axonal degenerative disorders.

Direct Tumor Killing and Immunotherapy through Anti-SerpinB9 Therapy.

Cancer therapies kill tumors either directly or indirectly by evoking immune responses and have been combined with varying levels of success. Here, we describe a paradigm to control cancer growth that is based on both direct tumor killing and the triggering of protective immunity. Genetic ablation of serine protease inhibitor SerpinB9 (Sb9) results in the death of tumor cells in a granzyme B (GrB)-dependent manner. Sb9-deficient mice exhibited protective T cell-based host immunity to tumors in association with a decline in GrB-expressing immunosuppressive cells within the tumor microenvironment (TME). Maximal protection against tumor development was observed when the tumor and host were deficient in Sb9. The therapeutic utility of Sb9 inhibition was demonstrated by the control of tumor growth, resulting in increased survival times in mice. Our studies describe a molecular target that permits a combination of tumor ablation, interference within the TME, and immunotherapy in one potential modality.

The nucleosome acidic patch and H2A ubiquitination underlie mSWI/SNF recruitment in synovial sarcoma.

Interactions between chromatin-associated proteins and the histone landscape play major roles in dictating genome topology and gene expression. Cancer-specific fusion oncoproteins, which display unique chromatin localization patterns, often lack classical DNA-binding domains, presenting challenges in identifying mechanisms governing their site-specific chromatin targeting and function. Here we identify a minimal region of the human SS18-SSX fusion oncoprotein (the hallmark driver of synovial sarcoma) that mediates a direct interaction between the mSWI/SNF complex and the nucleosome acidic patch. This binding results in altered mSWI/SNF composition and nucleosome engagement, driving cancer-specific mSWI/SNF complex targeting and gene expression. Furthermore, the C-terminal region of SSX confers preferential affinity to repressed, H2AK119Ub-marked nucleosomes, underlying the selective targeting to polycomb-marked genomic regions and synovial sarcoma-specific dependency on PRC1 function. Together, our results describe a functional interplay between a key nucleosome binding hub and a histone modification that underlies the disease-specific recruitment of a major chromatin remodeling complex.

Identification of a potent and selective covalent Pin1 inhibitor.

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is commonly overexpressed in human cancers, including pancreatic ductal adenocarcinoma (PDAC). While Pin1 is dispensable for viability in mice, it is required for activated Ras to induce tumorigenesis, suggesting a role for Pin1 inhibitors in Ras-driven tumors, such as PDAC. We report the development of rationally designed peptide inhibitors that covalently target Cys113, a highly conserved cysteine located in the Pin1 active site. The inhibitors were iteratively optimized for potency, selectivity and cell permeability to give BJP-06-005-3, a versatile tool compound with which to probe Pin1 biology and interrogate its role in cancer. In parallel to inhibitor development, we employed genetic and chemical-genetic strategies to assess the consequences of Pin1 loss in human PDAC cell lines. We demonstrate that Pin1 cooperates with mutant KRAS to promote transformation in PDAC, and that Pin1 inhibition impairs cell viability over time in PDAC cell lines.