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In 'Reconciling East and West' (December 2008) Richard John Neuhaus rightfully laments the remaining division--so close and yet so far away--that separates Christians East and West. Not long after converting [...]

Immunomodulatory imide drugs (IMiDs) are central components of therapy for multiple myeloma (MM). IMiDs bind cereblon (CRBN), an adaptor for the CUL4-DDB1-RBX1 E3 ligase to change its substrate specificity and induce degradation of 'neosubstrate' transcription factors that are essential to MM cells. Mechanistic studies to date have largely focussed on mediators of therapeutic activity and insight into clinical IMiD toxicities is less developed. We adopted BioID2-dependent proximity labelling (BioID2-CRBN) to characterise the CRBN interactome in the presence and absence of various IMiDs and the proteasome inhibitor, bortezomib. We aimed to leverage this technology to further map CRBN interactions beyond what has been achieved by conventional proteomic techniques. In support of this approach, analysis of cells expressing BioID2-CRBN following IMiD treatment displayed biotinylation of known CRBN interactors and neosubstrates. We observed that bortezomib alone significantly modifies the CRBN interactome. Proximity labelling also suggested that IMiDs augment the interaction between CRBN and proteins that are not degraded, thus designating 'neointeractors' distinct from previously disclosed 'neosubstrates'. Here we identify Non-Muscle Myosin Heavy Chain IIA (MYH9) as a putative CRBN neointeractor that may contribute to the haematological toxicity of IMiDs. These studies provide proof of concept for proximity labelling technologies in the mechanistic profiling of IMiDs and related E3-ligase-modulating drugs., (© 2024 The Author(s). The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

It has been widely appreciated that numerous bacterial species express chitinases for the purpose of degrading environmental chitin. However, chitinases and chitin-binding proteins are also expressed by pathogenic bacterial species during infection even though mammals do not produce chitin. Alternative molecular targets are therefore likely present within the host. Here, we will describe our current understanding of chitinase/chitin-binding proteins as virulence factors that promote bacterial colonization and infection. The targets of these chitinases in the host have been shown to include immune system components, mucins, and surface glycans. Bacterial chitinases have also been shown to interact with other microorganisms, targeting the peptidoglycan or chitin in the bacterial and fungal cell wall, respectively. This review highlights that even though the name "chitinase" implies activity toward chitin, chitinases can have a wide diversity of targets, including ones relevant to host infection. Chitinases may therefore be useful as a target of future anti-infective therapeutics., Competing Interests: The authors declare no conflict of interest.

Preclinical tumor models with native tissue microenvironments provide essential tools to understand how heterogeneous tumor phenotypes relate to drug response. Here we present syngeneic graft models of aggressive, metastasis-prone histopathology-specific NSCLC tumor types driven by KRAS mutation and loss of LKB1 (KL): adenosquamous carcinoma (ASC) and adenocarcinoma (AC). We show that subcutaneous injection of primary KL; ASC cells results in squamous cell carcinoma (SCC) tumors with high levels of stromal infiltrates, lacking the source heterogeneous histotype. Despite forming subcutaneous tumors, intravenously injected KL;AC cells were unable to form lung tumors. In contrast, intravenous injection of KL;ASC cells leads to their lung re-colonization and lesions recapitulating the mixed AC and SCC histopathology, tumor immune suppressive microenvironment and oncogenic signaling profile of source tumors, demonstrating histopathology-selective phenotypic dominance over genetic drivers. Pan-ERBB inhibition increased survival, while selective ERBB1/EGFR inhibition did not, suggesting a role of the ERBB network crosstalk in resistance to ERBB1/EGFR. This immunocompetent NSCLC lung colonization model hence phenocopies key properties of the metastasis-prone ASC histopathology, and serves as a preclinical model to dissect therapy responses and metastasis-associated processes., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The fungal gut microbiota (mycobiota) has been implicated in diseases that disturb gut homeostasis, such as inflammatory bowel disease. However, little is known about functional relationships between bacteria and fungi in the gut during infectious colitis. Here we investigated the role of fungal metabolites during infection with the intestinal pathogen Salmonella enterica serovar Typhimurium, a major cause of gastroenteritis worldwide. We found that, in the gut lumen, both the mycobiota and fungi present in the diet can be a source of siderophores, small molecules that scavenge iron from the host. The ability to use fungal siderophores, such as ferrichrome and coprogen, conferred a competitive growth advantage to Salmonella strains expressing the fungal siderophore receptors FhuA or FhuE in vitro and in a mouse model. Our study highlights the role of inter-kingdom cross-feeding between fungi and Salmonella and elucidates an additional function of the gut mycobiota, revealing the importance of these understudied members of the gut ecosystem during bacterial infection., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

MHC restriction, which describes the binding of TCRs from CD4 + T cells to class II MHC proteins and TCRs from CD8 + T cells to class I MHC proteins, is a hallmark of immunology. Seemingly rare TCRs that break this paradigm exist, but mechanistic insight into their behavior is lacking. TIL1383I is a prototypical class-mismatched TCR, cloned from a CD4 + T cell but recognizing the tyrosinase tumor antigen presented by the class I MHC HLA-A2 in a fully functional manner. Here we find that TIL1383I binds this class I target with a highly atypical geometry. Despite unorthodox binding, TCR signaling, antigen specificity, and the ability to use CD8 are maintained. Structurally, a key feature of TIL1383I is an exceptionally long CDR3β loop that mediates functions that are traditionally performed separately by hypervariable and germline loops in canonical TCR structures. Our findings thus expand the range of known TCR binding geometries compatible with normal function and specificity, provide insight into the determinants of MHC restriction, and may help guide TCR selection and engineering for immunotherapy., (© 2022. The Author(s).)

Salmonella enterica serovar Typhimurium (S. Typhimurium) is one of the leading causes of food-borne illnesses worldwide. To colonize the gastrointestinal tract, S. Typhimurium produces multiple virulence factors that facilitate cellular invasion. Chitinases have been recently emerging as virulence factors for various pathogenic bacterial species, and the S. Typhimurium genome contains two annotated chitinases: STM0018 (chiA) and STM0233. However, the role of these chitinases during S. Typhimurium pathogenesis is unknown. The putative chitinase STM0233 has not been studied previously, and only limited data exists on ChiA. Chitinases typically hydrolyze chitin polymers, which are absent in vertebrates. However, chiA expression was detected in infection models and purified ChiA cleaved carbohydrate subunits present on mammalian surface glycoproteins, indicating a role during pathogenesis. Here, we demonstrate that expression of chiA and STM0233 is upregulated in the mouse gut and that both chitinases facilitate epithelial cell adhesion and invasion. S. Typhimurium lacking both chitinases showed a 70% reduction in invasion of small intestinal epithelial cells in vitro. In a gastroenteritis mouse model, chitinase-deficient S. Typhimurium strains were also significantly attenuated in the invasion of small intestinal tissue. This reduced invasion resulted in significantly delayed S. Typhimurium dissemination to the spleen and the liver, but chitinases were not required for systemic survival. The invasion defect of the chitinase-deficient strain was rescued by the presence of wild-type S. Typhimurium, suggesting that chitinases are secreted. By analyzing N-linked glycans of small intestinal cells, we identified specific N-acetylglucosamine-containing glycans as potential extracellular targets of S. Typhimurium chitinases. This analysis also revealed a differential abundance of Lewis X/A-containing glycans that is likely a result of host cell modulation due to the detection of S. Typhimurium chitinases. Similar glycomic changes elicited by chitinase deficient strains indicate functional redundancy of the chitinases. Overall, our results demonstrate that S. Typhimurium chitinases contribute to intestinal adhesion and invasion through modulation of the host glycome., Competing Interests: The authors have declared that no competing interests exist.

Accurate control of gene expression is essential for normal development and dysregulation of transcription underpins cancer onset and progression. Similar to cell cycle regulation, RNA polymerase II-driven transcription can be considered as a unidirectional multistep cycle, with thousands of unique transcription cycles occurring in concert within each cell. Each transcription cycle comprises recruitment, initiation, pausing, elongation, termination and recycling stages that are tightly controlled by the coordinated action of transcriptional cyclin-dependent kinases and their cognate cyclins as well as the opposing activity of transcriptional phosphatases. Oncogenic dysregulation of transcription can entail defective control of gene expression, either at select loci or more globally, impacting a large proportion of the genome. The resultant dependency on the core-transcriptional machinery is believed to render 'transcriptionally addicted' cancers sensitive to perturbation of transcription. Based on these findings, small molecules targeting transcriptional cyclin-dependent kinases and associated proteins hold promise for the treatment of cancer. Here, we utilize the transcription cycles concept to explain how dysregulation of these finely tuned gene expression processes may drive tumorigenesis and how therapeutically beneficial responses may arise from global or selective transcriptional perturbation. This conceptual framework helps to explain tumour-selective transcriptional dependencies and facilitates the rational design of combination therapies., (© 2021. Springer Nature Limited.)

Potassium ion homeostasis is essential for bacterial survival, playing roles in osmoregulation, pH homeostasis, regulation of protein synthesis, enzyme activation, membrane potential adjustment and electrical signaling. To accomplish such diverse physiological tasks, it is not surprising that a single bacterium typically encodes several potassium uptake and release systems. To understand the role each individual protein fulfills and how these proteins work in concert, it is important to identify the molecular details of their function. One needs to understand whether the systems transport ions actively or passively, and what mechanisms or ligands lead to the activation or inactivation of individual systems. Combining mechanistic information with knowledge about the physiology under different stress situations, such as osmostress, pH stress or nutrient limitation, one can identify the task of each system and deduce how they are coordinated with each other. By reviewing the general principles of bacterial membrane physiology and describing the molecular architecture and function of several bacterial K + -transporting systems, we aim to provide a framework for microbiologists studying bacterial potassium homeostasis and the many K + -translocating systems that are still poorly understood., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Gene expression by RNA polymerase II (RNAPII) is tightly controlled by cyclin-dependent kinases (CDKs) at discrete checkpoints during the transcription cycle. The pausing checkpoint following transcription initiation is primarily controlled by CDK9. We discovered that CDK9-mediated, RNAPII-driven transcription is functionally opposed by a protein phosphatase 2A (PP2A) complex that is recruited to transcription sites by the Integrator complex subunit INTS6. PP2A dynamically antagonizes phosphorylation of key CDK9 substrates including DSIF and RNAPII-CTD. Loss of INTS6 results in resistance to tumor cell death mediated by CDK9 inhibition, decreased turnover of CDK9 phospho-substrates, and amplification of acute oncogenic transcriptional responses. Pharmacological PP2A activation synergizes with CDK9 inhibition to kill both leukemic and solid tumor cells, providing therapeutic benefit in vivo. These data demonstrate that fine control of gene expression relies on the balance between kinase and phosphatase activity throughout the transcription cycle, a process dysregulated in cancer that can be exploited therapeutically., Competing Interests: Declaration of interests The Johnstone laboratory receives funding support from Roche, BMS, Astra Zeneca, and MecRx. R.W.J. is a shareholder in MecRx and receives consultancy payments., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Internal tandem duplication of the FMS-like tyrosine kinase 3 gene ( FLT3-ITD ) occurs in 30% of all acute myeloid leukemias (AML). Limited clinical efficacy of FLT3 inhibitors highlights the need for alternative therapeutic modalities in this subset of disease. Using human and murine models of FLT3-ITD-driven AML, we demonstrate that FLT3-ITD promotes serine synthesis and uptake via ATF4-dependent transcriptional regulation of genes in the de novo serine biosynthesis pathway and neutral amino acid transport. Genetic or pharmacologic inhibition of PHGDH, the rate-limiting enzyme of de novo serine biosynthesis, selectively inhibited proliferation of FLT3-ITD AMLs in vitro and in vivo . Moreover, pharmacologic inhibition of PHGDH sensitized FLT3-ITD AMLs to the standard-of-care chemotherapeutic cytarabine. Collectively, these data reveal novel insights into FLT3-ITD-induced metabolic reprogramming and reveal a targetable vulnerability in FLT3-ITD AML. SIGNIFICANCE: FLT3-ITD mutations are common in AML and are associated with poor prognosis. We show that FLT3-ITD stimulates serine biosynthesis, thereby rendering FLT3-ITD-driven leukemias dependent upon serine for proliferation and survival. This metabolic dependency can be exploited pharmacologically to sensitize FLT3-ITD-driven AMLs to chemotherapy. This article is highlighted in the In This Issue feature, p. 1307 ., (©2021 American Association for Cancer Research.)

To separate causal effects of histone acetylation on chromatin accessibility and transcriptional output, we used integrated epigenomic and transcriptomic analyses following acute inhibition of major cellular lysine acetyltransferases P300 and CBP in hematological malignancies. We found that catalytic P300/CBP inhibition dynamically perturbs steady-state acetylation kinetics and suppresses oncogenic transcriptional networks in the absence of changes to chromatin accessibility. CRISPR-Cas9 screening identified NCOR1 and HDAC3 transcriptional co-repressors as the principal antagonists of P300/CBP by counteracting acetylation turnover kinetics. Finally, deacetylation of H3K27 provides nucleation sites for reciprocal methylation switching, a feature that can be exploited therapeutically by concomitant KDM6A and P300/CBP inhibition. Overall, this study indicates that the steady-state histone acetylation-methylation equilibrium functions as a molecular rheostat governing cellular transcription that is amenable to therapeutic exploitation as an anti-cancer regimen., Competing Interests: Declaration of interests The Johnstone laboratory receives funding support from Roche, Bristol Myers Squibb (BMS), AstraZeneca, and MecRx. R.W.J. is a shareholder in and consultant for MecRx. A.L. and K.D.B. are employees of and shareholders in AbbVie. A.S. has participated on advisory boards for and received research funding from Celgene, Juno, BMS, Janssen-Cilag, Novartis, Amgen, Haemalogix, Abbvie, and Takeda. J.S. has participated on advisory boards for and received honoraria from Celgene. O.A.-W. has served as a consultant for H3B Biomedicine, Foundation Medicine Inc., Merck, Prelude Therapeutics, and Janssen; is on the scientific advisory boards of Envisagenics Inc., Pfizer Boulder, and AIChemy Inc.; and has received prior research funding from Loxo Oncology and H3 Biomedicine. J.D.L. is a scientific adviser to the Samuel Waxman Cancer Research Foundation. All other authors declare no competing interests., (Crown Copyright © 2021. Published by Elsevier Inc. All rights reserved.)

The term "microbiota" invokes images of mucosal surfaces densely populated with bacteria. These surfaces and the luminal compartments they form indeed predominantly harbor bacteria. However, research from this past decade has started to complete the picture by focusing on important but largely neglected constituents of the microbiota: fungi, viruses, and archaea. The community of commensal fungi, also called the mycobiota, interacts with commensal bacteria and the host. It is thus not surprising that changes in the mycobiota have significant impact on host health and are associated with pathological conditions such as inflammatory bowel disease (IBD). In this review we will give an overview of why the mycobiota is an important research area and different mycobiota research tools. We will specifically focus on distinguishing transient and actively colonizing fungi of the oral and gut mycobiota and their roles in health and disease. In addition to correlative and observational studies, we will discuss mechanistic studies on specific cross-kingdom interactions of fungi, bacteria, and the host., (Copyright © 2021 Santus et al.)

CD8+ T cell recognition of peptide epitopes plays a central role in immune responses against pathogens and tumors. However, the rules that govern which peptides are truly recognized by existing T cell receptors (TCRs) remain poorly understood, precluding accurate predictions of neo-epitopes for cancer immunotherapy. Here, we capitalize on recent (neo-)epitope data to train a predictor of immunogenic epitopes (PRIME), which captures molecular properties of both antigen presentation and TCR recognition. PRIME not only improves prioritization of neo-epitopes but also correlates with T cell potency and unravels biophysical determinants of TCR recognition that we experimentally validate. Analysis of cancer genomics data reveals that recurrent mutations tend to be less frequent in patients where they are predicted to be immunogenic, providing further evidence for immunoediting in human cancer. PRIME will facilitate identification of pathogen epitopes in infectious diseases and neo-epitopes in cancer immunotherapy., Competing Interests: The authors declare no competing interests. G. Ciriello is a member of the advisory board of Cell Reports Medicine., (© 2021 The Author(s).)

Using a variety of activating and inhibitory receptors, natural killer (NK) cells protect against disease by eliminating cells that have downregulated class I major histocompatibility complex (MHC) proteins, such as in response to cell transformation or viral infection. The inhibitory murine NK receptor Ly49C specifically recognizes the class I MHC protein H-2K b . Unusual among NK receptors, Ly49C exhibits a peptide-dependent sensitivity to H-2K b recognition, which has not been explained despite detailed structural studies. To gain further insight into Ly49C peptide sensitivity, we examined Ly49C recognition biochemically and through the lens of dynamic allostery. We found that the peptide sensitivity of Ly49C arises through small differences in H-2K b -binding affinity. Although molecular dynamics simulations supported a role for peptide-dependent protein dynamics in producing these differences in binding affinity, calorimetric measurements indicated an enthalpically as opposed to entropically driven process. A quantitative linkage analysis showed that this emerges from peptide-dependent dynamic tuning of electrostatic interactions across the Ly49C-H-2K b interface. We propose a model whereby different peptides alter the flexibility of H-2K b , which in turn changes the strength of electrostatic interactions across the protein-protein interface. Our results provide a quantitative assessment of how peptides alter Ly49C-binding affinity, suggest the underlying mechanism, and demonstrate peptide-driven allostery at work in class I MHC proteins. Lastly, our model provides a solution for how dynamic allostery could impact binding of some, but not all, class I MHC partners depending on the structural and chemical composition of the interfaces., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Elevated ribosome biogenesis in oncogene-driven cancers is commonly targeted by DNA-damaging cytotoxic drugs. Our previous first-in-human trial of CX-5461, a novel, less genotoxic agent that specifically inhibits ribosome biogenesis via suppression of RNA polymerase I (Pol I) transcription, revealed single-agent efficacy in refractory blood cancers. Despite this clinical response, patients were not cured. In parallel, we demonstrated a marked improvement in the in vivo efficacy of CX-5461 in combination with PI3K/AKT/mTORC1 pathway inhibitors. Here, we reveal the molecular basis for this improved efficacy observed in vivo, which is associated with specific suppression of translation of mRNAs encoding regulators of cellular metabolism. Importantly, acquired resistance to this cotreatment is driven by translational rewiring that results in dysregulated cellular metabolism and induction of a cAMP-dependent pathway critical for the survival of blood cancers including lymphoma and acute myeloid leukemia. Our studies thus identify key molecular mechanisms underpinning the response of blood cancers to selective inhibition of ribosome biogenesis and define metabolic vulnerabilities that will facilitate the rational design of more effective regimens for Pol I-directed therapies., (© 2020 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

T-cell recognition of peptides incorporating nonsynonymous mutations, or neoepitopes, is a cornerstone of tumor immunity and forms the basis of new immunotherapy approaches including personalized cancer vaccines. Yet as they are derived from self-peptides, the means through which immunogenic neoepitopes overcome immune self-tolerance are often unclear. Here we show that a point mutation in a non-major histocompatibility complex anchor position induces structural and dynamic changes in an immunologically active ovarian cancer neoepitope. The changes pre-organize the peptide into a conformation optimal for recognition by a neoepitope-specific T-cell receptor, allowing the receptor to bind the neoepitope with high affinity and deliver potent T-cell signals. Our results emphasize the importance of structural and physical changes relative to self in neoepitope immunogenicity. Considered broadly, these findings can help explain some of the difficulties in identifying immunogenic neoepitopes from sequence alone and provide guidance for developing novel, neoepitope-based personalized therapies.

MCL-1 is one of the most frequently amplified genes in cancer, facilitating tumor initiation and maintenance and enabling resistance to anti-tumorigenic agents including the BCL-2 selective inhibitor venetoclax. The expression of MCL-1 is maintained via P-TEFb-mediated transcription, where the kinase CDK9 is a critical component. Consequently, we developed a series of potent small-molecule inhibitors of CDK9, exemplified by the orally active A-1592668, with CDK selectivity profiles that are distinct from related molecules that have been extensively studied clinically. Short-term treatment with A-1592668 rapidly downregulates RNA pol-II (Ser 2) phosphorylation resulting in the loss of MCL-1 protein and apoptosis in MCL-1-dependent hematologic tumor cell lines. This cell death could be attenuated by either inhibiting caspases or overexpressing BCL-2 protein. Synergistic cell killing was also observed between A-1592668 or the related analog A-1467729, and venetoclax in a number of hematologic cell lines and primary NHL patient samples. Importantly, the CDK9 inhibitor plus venetoclax combination was well tolerated in vivo and demonstrated efficacy superior to either agent alone in mouse models of lymphoma and AML. These data indicate that CDK9 inhibitors could be highly efficacious in tumors that depend on MCL-1 for survival or when used in combination with venetoclax in malignancies dependent on MCL-1 and BCL-2.

The RNA polymerase II (POLII)-driven transcription cycle is tightly regulated at distinct checkpoints by cyclin-dependent kinases (CDKs) and their cognate cyclins. The molecular events underpinning transcriptional elongation, processivity, and the CDK-cyclin pair(s) involved remain poorly understood. Using CRISPR-Cas9 homology-directed repair, we generated analog-sensitive kinase variants of CDK12 and CDK13 to probe their individual and shared biological and molecular roles. Single inhibition of CDK12 or CDK13 induced transcriptional responses associated with cellular growth signaling pathways and/or DNA damage, with minimal effects on cell viability. In contrast, dual kinase inhibition potently induced cell death, which was associated with extensive genome-wide transcriptional changes including widespread use of alternative 3' polyadenylation sites. At the molecular level, dual kinase inhibition resulted in the loss of POLII CTD phosphorylation and greatly reduced POLII elongation rates and processivity. These data define substantial redundancy between CDK12 and CDK13 and identify both as fundamental regulators of global POLII processivity and transcription elongation., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Most non-small cell lung cancers (NSCLC) contain nontargetable mutations, including KRAS, TP53 , or STK11/LKB1 alterations. By coupling ex vivo drug sensitivity profiling with in vivo drug response studies, we aimed to identify drug vulnerabilities for these NSCLC subtypes. Primary adenosquamous carcinoma (ASC) or adenocarcinoma (AC) cultures were established from Kras G12D/+ ;Lkb1 fl/fl (KL) tumors or AC cultures from Kras G12D/+ ;p53 fl/fl (KP) tumors. Although p53 -null cells readily propagated as conventional cultures, Lkb1 -null cells required conditional reprograming for establishment. Drug response profiling revealed short-term response to MEK inhibition, yet long-term clonogenic assays demonstrated resistance, associated with sustained or adaptive activation of receptor tyrosine kinases (RTK): activation of ERBBs in KL cultures, or FGFR in AC cultures. Furthermore, pan-ERBB inhibition reduced the clonogenicity of KL cultures, which was exacerbated by combinatorial MEK inhibition, whereas combinatorial MEK and FGFR inhibition suppressed clonogenicity of AC cultures. Importantly, in vivo studies confirmed KL-selective sensitivity to pan-ERBB inhibition, which correlated with high ERBB ligand expression and activation of ERBB receptors, implying that ERBB network activity may serve as a predictive biomarker of drug response. Interestingly, in human NSCLCs, phosphorylation of EGFR or ERBB3 was frequently detected in ASCs and squamous cell carcinomas. We conclude that analysis of in situ ERBB signaling networks in conjunction with ex vivo drug response profiling and biochemical dissection of adaptive RTK activities may serve as a valid diagnostic approach to identify tumors sensitive to ERBB network inhibition., (©2019 American Association for Cancer Research.)

The development of immunological therapies that incorporate peptide antigens presented to T cells by MHC proteins is a long sought-after goal, particularly for cancer, where mutated neoantigens are being explored as personalized cancer vaccines. Although neoantigens can be identified through sequencing, bioinformatics and mass spectrometry, identifying those which are immunogenic and able to promote tumor rejection remains a significant challenge. Here we examined the potential of high-resolution structural modeling followed by energetic scoring of structural features for predicting neoantigen immunogenicity. After developing a strategy to rapidly and accurately model nonameric peptides bound to the common class I MHC protein HLA-A2, we trained a neural network on structural features that influence T cell receptor (TCR) and peptide binding energies. The resulting structurally-parameterized neural network outperformed methods that do not incorporate explicit structural or energetic properties in predicting CD8 + T cell responses of HLA-A2 presented nonameric peptides, while also providing insight into the underlying structural and biophysical mechanisms governing immunogenicity. Our proof-of-concept study demonstrates the potential for structure-based immunogenicity predictions in the development of personalized peptide-based vaccines.

T cell receptors (TCRs) have emerged as a new class of immunological therapeutics. However, though antigen specificity is a hallmark of adaptive immunity, TCRs themselves do not possess the high specificity of monoclonal antibodies. Although a necessary function of T cell biology, the resulting cross-reactivity presents a significant challenge for TCR-based therapeutic development, as it creates the potential for off-target recognition and immune toxicity. Efforts to enhance TCR specificity by mimicking the antibody maturation process and enhancing affinity can inadvertently exacerbate TCR cross-reactivity. Here we demonstrate this concern by showing that even peptide-targeted mutations in the TCR can introduce new reactivities against peptides that bear similarity to the original target. To counteract this, we explored a novel structure-guided approach for enhancing TCR specificity independent of affinity. Tested with the MART-1-specific TCR DMF5, our approach had a small but discernible impact on cross-reactivity toward MART-1 homologs yet was able to eliminate DMF5 cross-recognition of more divergent, unrelated epitopes. Our study provides a proof of principle for the use of advanced structure-guided design techniques for improving TCR specificity, and it suggests new ways forward for enhancing TCRs for therapeutic use., (Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Despite the development of novel drugs, the prospects for many patients with acute myeloid leukemia (AML) remain dismal. This study reveals that the selective inhibitor of RNA polymerase I (Pol I) transcription, CX-5461, effectively treats aggressive AML, including mixed-lineage leukemia-driven AML, and outperforms standard chemotherapies. In addition to the previously characterized mechanism of action of CX-5461 (ie, the induction of p53-dependent apoptotic cell death), the inhibition of Pol I transcription also demonstrates potent efficacy in p53null AML in vivo. This significant survival advantage in both p53WT and p53null leukemic mice treated with CX-5461 is associated with activation of the checkpoint kinases 1/2, an aberrant G2/M cell-cycle progression and induction of myeloid differentiation of the leukemic blasts. The ability to target the leukemic-initiating cell population is thought to be essential for lasting therapeutic benefit. Most strikingly, the acute inhibition of Pol I transcription reduces both the leukemic granulocyte-macrophage progenitor and leukemia-initiating cell (LIC) populations, and suppresses their clonogenic capacity. This suggests that dysregulated Pol I transcription is essential for the maintenance of their leukemia-initiating potential. Together, these findings demonstrate the therapeutic utility of this new class of inhibitors to treat highly aggressive AML by targeting LICs., (© 2017 by The American Society of Hematology.)

Lung cancers exhibit pronounced functional heterogeneity, confounding precision medicine. We studied how the cell of origin contributes to phenotypic heterogeneity following conditional expression of Kras G12D and loss of Lkb1 (Kras;Lkb1). Using progenitor cell-type-restricted adenoviral Cre to target cells expressing surfactant protein C (SPC) or club cell antigen 10 (CC10), we show that Ad5-CC10-Cre-infected mice exhibit a shorter latency compared with Ad5-SPC-Cre cohorts. We further demonstrate that CC10 + cells are the predominant progenitors of adenosquamous carcinoma (ASC) tumors and give rise to a wider spectrum of histotypes that includes mucinous and acinar adenocarcinomas. Transcriptome analysis shows ASC histotype-specific upregulation of pro-inflammatory and immunomodulatory genes. This is accompanied by an ASC-specific immunosuppressive environment, consisting of downregulated MHC genes, recruitment of CD11b + Gr-1 + tumor-associated neutrophils (TANs), and decreased T cell numbers. We conclude that progenitor cell-specific etiology influences the Kras;Lkb1-driven tumor histopathology spectrum and histotype-specific immune microenvironment., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: The MYC oncogene is frequently overexpressed in prostate cancer. Upregulation of ribosome biogenesis and function is characteristic of MYC-driven tumors. In addition, PIM kinases activate MYC signaling and mRNA translation in prostate cancer and cooperate with MYC to accelerate tumorigenesis. Here, we investigate the efficacy of a single and dual approach targeting ribosome biogenesis and function to treat prostate cancer., Experimental Design: The inhibition of ribosomal RNA (rRNA) synthesis with CX-5461, a potent, selective, and orally bioavailable inhibitor of RNA polymerase I (Pol I) transcription, has been successfully exploited therapeutically but only in models of hematologic malignancy. CX-5461 and CX-6258, a pan-PIM kinase inhibitor, were tested alone and in combination in prostate cancer cell lines, in Hi-MYC- and PTEN-deficient mouse models and in patient-derived xenografts (PDX) of metastatic tissue obtained from a patient with castration-resistant prostate cancer., Results: CX-5461 inhibited anchorage-independent growth and induced cell-cycle arrest in prostate cancer cell lines at nanomolar concentrations. Oral administration of 50 mg/kg CX-5461 induced TP53 expression and activity and reduced proliferation (MKI67) and invasion (loss of ductal actin) in Hi-MYC tumors, but not in PTEN-null (low MYC) tumors. While 100 mg/kg CX-6258 showed limited effect alone, its combination with CX-5461 further suppressed proliferation and dramatically reduced large invasive lesions in both models. This rational combination strategy significantly inhibited proliferation and induced cell death in PDX of prostate cancer., Conclusions: Our results demonstrate preclinical efficacy of targeting the ribosome at multiple levels and provide a new approach for the treatment of prostate cancer. Clin Cancer Res; 22(22); 5539-52. ©2016 AACR., (©2016 American Association for Cancer Research.)

RNA polymerase I (Pol I)-mediated transcription of the ribosomal RNA genes (rDNA) is confined to the nucleolus and is a rate-limiting step for cell growth and proliferation. Inhibition of Pol I by CX-5461 can selectively induce p53-mediated apoptosis of tumour cells in vivo. Currently, CX-5461 is in clinical trial for patients with advanced haematological malignancies (Peter Mac, Melbourne). Here we demonstrate that CX-5461 also induces p53-independent cell cycle checkpoints mediated by ATM/ATR signaling in the absence of DNA damage. Further, our data demonstrate that the combination of drugs targeting ATM/ATR signaling and CX-5461 leads to enhanced therapeutic benefit in treating p53-null tumours in vivo, which are normally refractory to each drug alone. Mechanistically, we show that CX-5461 induces an unusual chromatin structure in which transcriptionally competent relaxed rDNA repeats are devoid of transcribing Pol I leading to activation of ATM signaling within the nucleoli. Thus, we propose that acute inhibition of Pol transcription initiation by CX-5461 induces a novel nucleolar stress response that can be targeted to improve therapeutic efficacy., Competing Interests: D. Drygin is VP (R&D) of Pimera Inc., San Diego, CA, USA. R.D. Hannan, R.B. Pearson and G.A. McArthur are Scientific Advisors to Pimera Inc. R.W. Johnstone has a commercial research grant and has received honoraria for service on the speakers' bureau for Novartis. G.A. McArthur has commercial research grants from Celgene and Pfizer. No potential conflicts of interest were disclosed by the other authors.

Unlabelled: Ribosome biogenesis and protein synthesis are dysregulated in many cancers, with those driven by the proto-oncogene c-MYC characterized by elevated Pol I-mediated ribosomal rDNA transcription and mTORC1/eIF4E-driven mRNA translation. Here, we demonstrate that coordinated targeting of rDNA transcription and PI3K-AKT-mTORC1-dependent ribosome biogenesis and protein synthesis provides a remarkable improvement in survival in MYC-driven B lymphoma. Combining an inhibitor of rDNA transcription (CX-5461) with the mTORC1 inhibitor everolimus more than doubled survival of Eμ-Myc lymphoma-bearing mice. The ability of each agent to trigger tumor cell death via independent pathways was central to their synergistic efficacy. CX-5461 induced nucleolar stress and p53 pathway activation, whereas everolimus induced expression of the proapoptotic protein BMF that was independent of p53 and reduced expression of RPL11 and RPL5. Thus, targeting the network controlling the synthesis and function of ribosomes at multiple points provides a potential new strategy to treat MYC-driven malignancies., Significance: Treatment options for the high proportion of cancers driven by MYC are limited. We demonstrate that combining pharmacologic targeting of ribosome biogenesis and mTORC1-dependent translation provides a remarkable therapeutic benefit to Eμ-Myc lymphoma-bearing mice. These results establish a rationale for targeting ribosome biogenesis and function to treat MYC-driven cancer., (©2015 American Association for Cancer Research.)

Six Zn(II) complexes of derivatives of 1,4,7,10-tetraazacyclododecane (cyclen) were studied for binding to DNA sequences containing non-canonical thymines, including a hairpin with a single thymine bulge (T-bulge) and a G-quadruplex (H-telo) containing thymine loops. The cyclen-based macrocycles contained pendents with either two fused rings to give planar groups including quinolinone (QMC), coumarin (MCC) and quinoline (CQC) derivatives or a non-planar dansyl group (DSC). Macrocyclic complexes with three fused rings including an anthraquinone pendent (ATQ) were also studied. All Zn(II) complexes were stable in solution at micromolar concentrations and neutral pH with the Zn(L)(OH2) species prevailing for L = QMC and CQC at pH 7.5 and 100 mM NaCl. Immobilized T-bulge or H-telo G-quadruplex was used to study binding of the complexes by surface plasmon resonance (SPR) for several of the complexes. For the most part, data matched well with that obtained by isothermal calorimetry (ITC) and, for fluorescent complexes, by fluorescence titrations. Data showed that Zn(II) complexes containing planar aromatic pendents with two fused rings bound to T-bulge more tightly than complexes with non-planar pendents such as DSC. The H-telo DNA exhibited multiple binding sites for all complexes containing aromatic pendents. The complexes with two fused rings bound with low micromolar dissociation constants and two binding sites whereas a complex with three fused rings (ATQ) bound to three sites. This study shows that different pendent groups on Zn(II) cyclen complexes impart selectivity for recognition of non-canonical DNA structures.

The contribution of the nucleolus to cancer is well established with respect to its traditional role in facilitating ribosome biogenesis and proliferative capacity. More contemporary studies however, infer that nucleoli contribute a much broader role in malignant transformation. Specifically, extra-ribosomal functions of the nucleolus position it as a central integrator of cellular proliferation and stress signaling, and are emerging as important mechanisms for modulating how oncogenes and tumor suppressors operate in normal and malignant cells. The dependence of certain tumor cells to co-opt nucleolar processes to maintain their cancer phenotypes has now clearly been demonstrated by the application of small molecule inhibitors of RNA Polymerase I to block ribosomal DNA transcription and disrupt nucleolar function (Bywater et al., 2012 [1]). These drugs, which selectively kill tumor cells in vivo while sparing normal cells, have now progressed to clinical trials. It is likely that we have only just begun to scratch the surface of the potential of the nucleolus as a new target for cancer therapy, with "suppression of nucleolar stress" representing an emerging "hallmark" of cancer. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The dysregulation of PI3K/AKT/mTORC1 signalling and/or hyperactivation of MYC are observed in a high proportion of human cancers, and together they form a 'super signalling' network mediating malignancy. A fundamental downstream action of this signalling network is up-regulation of ribosome biogenesis and subsequent alterations in the patterns of translation and increased protein synthesis, which are thought to be critical for AKT/MYC-driven oncogenesis. We have demonstrated that AKT and MYC cooperate to drive ribosomal DNA (rDNA) transcription and ribosome biogenesis, with AKT being essential for rDNA transcription and in vitro survival of lymphoma cells isolated from a MYC-driven model of B-cell lymphoma (Eμ-Myc) [Chan JC et al., (2011) Science Signalling 4, ra56]. Here we show that the allosteric AKT inhibitor MK-2206 rapidly and potently antagonizes rDNA transcription in Eμ-Myc B-cell lymphomas in vivo, and this is associated with a rapid reduction in indicators of disease burden, including spleen weight and the abundance of tumour cells in both the circulation and lymph nodes. Extended treatment of tumour-bearing mice with MK-2206 resulted in a significant delay in disease progression, associated with increased B-cell lymphoma apoptosis. Our findings suggest that malignant diseases characterized by unrestrained ribosome biogenesis may be vulnerable to therapeutic strategies that target the PI3K/AKT/mTORC1/MYC growth control network., (© 2013 The Authors Journal compilation © 2013 FEBS.)

Pharmacological strategies capable of directly targeting MYC are elusive. Previous studies have shown that MYC-driven lymphomagenesis is associated with mammalian target of rapamycin (mTOR) activation and a MYC-evoked DNA damage response (DDR) transduced by phosphatidylinositol-3-kinase (PI3K)-related kinases (DNA-PK, ATM, and ATR). Here we report that BEZ235, a multitargeted pan-PI3K/dual-mTOR inhibitor, potently killed primary Myc-driven B-cell lymphomas and human cell lines bearing IG-cMYC translocations. Using pharmacologic and genetic dissection of PI3K/mTOR signaling, dual DDR/mTORC1 inhibition was identified as a key mediator of apoptosis. Moreover, apoptosis was initiated at drug concentrations insufficient to antagonize PI3K/mTORC2-regulated AKT phosphorylation. p53-independent induction of the proapoptotic BH3-only protein BMF was identified as a mechanism by which dual DDR/mTORC1 inhibition caused lymphoma cell death. BEZ235 treatment induced apoptotic tumor regressions in vivo that correlated with suppression of mTORC1-regulated substrates and reduced H2AX phosphorylation and also with feedback phosphorylation of AKT. These mechanistic studies hold important implications for the use of multitargeted PI3K inhibitors in the treatment of hematologic malignancies. In particular, the newly elucidated role of PI3K-related DDR kinases in response to PI3K inhibitors offers a novel therapeutic opportunity for the treatment of hematologic malignancies with an MYC-driven DDR.