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The article formulates the main aspects of understanding power as a social phenomenon. The philosophy of Power is a complex multi–faceted research project. Being a social phenomenon, power arises as a result of relations between people, and all means of power are determined by these relations. But there are those signs of power that reflect universal properties - subordination and dominance, the formation of subject–object relations, which form a power-subservient system.

Sickle cell disease (SCD) is a genetic disorder caused by a single point mutation (β6 Glu → Val) on the β-chain of adult hemoglobin (HbA) that results in sickled hemoglobin (HbS). In the deoxygenated state, polymerization of HbS leads to sickling of red blood cells (RBC). Several downstream consequences of polymerization and RBC sickling include vaso-occlusion, hemolytic anemia, and stroke. We report the design of a noncovalent modulator of HbS, clinical candidate PF-07059013 (23). The seminal hit molecule was discovered by virtual screening and confirmed through a series of biochemical and biophysical studies. After a significant optimization effort, we arrived at 23, a compound that specifically binds to Hb with nanomolar affinity and displays strong partitioning into RBCs. In a 2-week multiple dose study using Townes SCD mice, 23 showed a 37.8% (±9.0%) reduction in sickling compared to vehicle treated mice. 23 (PF-07059013) has advanced to phase 1 clinical trials.

The COVID-19 pandemic continues to be a public health threat with emerging variants of SARS-CoV-2. Nirmatrelvir (PF-07321332) is a reversible, covalent inhibitor targeting the main protease (M

The role of Fas in germinal center (GC) homeostasis is controversial. Razzaghi et al. show that Fas is a strong cell-intrinsic regulator of the GC and that its loss defines an aggressive subtype of GC-derived lymphoma., Fas is highly expressed on germinal center (GC) B cells, and mutations of FAS have been reported in diffuse large B cell lymphoma (DLBCL). Although GC-derived DLBCL has better overall outcomes than other DLBCL types, some cases are refractory, and the molecular basis for this is often unknown. We show that Fas is a strong cell-intrinsic regulator of GC B cells that promotes cell death in the light zone, likely via T follicular helper (Tfh) cell–derived Fas ligand. In the absence of Fas, GCs were more clonally diverse due to an accumulation of cells that did not demonstrably bind antigen. FAS alterations occurred most commonly in GC-derived DLBCL, were associated with inferior outcomes and an enrichment of Tfh cells, and co-occurred with deficiency in HVEM and PD-L1 that regulate the Tfh–B cell interaction. This work shows that Fas is critically required for GC homeostasis and suggests that loss of Tfh-mediated counterselection in the GC contributes to lethality in GC-derived lymphoma., Graphical Abstract

Purpose:We examined cabazitaxel, a novel next-generation taxoid, in patients with metastatic gastric cancer in a multicenter phase II study.Patients and Methods:Patients who have progressed on one or more prior therapies for locally advanced, unresectable, or metastatic disease were eligible, and prior taxane therapy was allowed. Taxane-naïve and pretreated cohorts were analyzed independently for efficacy. The primary endpoint for both cohorts was progression-free survival (PFS) using RECIST 1.1, using a Simon's two-stage design (10% significance and 80% power) for both cohorts. Comprehensive molecular annotation included whole exome and bulk RNA sequencing.Results:Fifty-three patients enrolled in the taxane-naïve cohort (Arm A) and 23 patients in the prior-taxane cohort (Arm B), from January 8, 2013, to April 8, 2015: median age 61.7 years (range, 35.5–91.8 years), 66% male, 66% Caucasian. The most common adverse events included neutropenia (17% Arm A and 39% Arm B), fatigue/muscle weakness (13%), and hematuria (12%). In Arm A, the 3-month PFS rate was 28% [95% confidence interval (CI), 17%–42%] and did not meet the prespecified efficacy target. The 3-month PFS rate in Arm B was 35% (95% CI, 16%–57%) and surpassed its efficacy target. HER2 amplification or overexpression was associated with improved disease control (P = 0.003), PFS (P = 0.04), and overall survival (P = 0.002). An M2 macrophage signature was also associated with improved survival (P = 0.031).Conclusions:Cabazitaxel has modest activity in advanced gastric cancer, including in patients previously treated with taxanes. Her2 amplification/overexpression and M2 high macrophage signature are potential biomarkers for taxane efficacy that warrant further evaluation.

Utilization of kidneys from extended criteria donors leads to an increase in average warm ischemia time (WIT), which is associated with larger degrees of ischemia-reperfusion injury (IRI). Kidney resuscitation by extracorporeal perfusion in situ allows up to 60 minutes of asystole after the circulatory death. Molecular studies of kidney grafts from human donors with critically expanded WIT are warranted. Transcriptomes of two human kidneys from two different donors were profiled after 35-45 minutes of WIT and after 120 minutes of normothermic perfusion and compared. Baseline gene expression patterns in ischemic grafts display substantial intrinsic differences. IRI does not lead to substantial change in overall transcription landscape but activates a highly connected protein network with hubs centered on Jun/Fos/ATF transcription factors and HSP1A/HSPA5 heat shock proteins. This response is regulated by positive feedback. IRI networks are enriched in soluble proteins and biofluids assayable substances, thus, indicating feasibility of the longitudinal, minimally invasive assessment in vivo. Mapping of IRI related molecules in ischemic and reperfused kidneys provides a rationale for possible organ conditioning during machine assisted ex vivo normothermic perfusion. A study of natural diversity of the transcriptional landscapes in presumably normal, transplantation-suitable human organs is warranted.

miRNAs play a key role in regulation of gene expression. Nowadays it is known more than 2500 human miRNAs, while a majority of miRNA–mRNA interactions remains unidentified. The recent development of a high-throughput CLASH (crosslinking, ligation and sequencing of hybrids) technique for discerning miRNA–mRNA interactions allowed an experimental analysis of the human miRNA–mRNA interactome. Therefore, it allowed us, for the first time, make an experimental analysis of the human miRNA–mRNA interactome as a whole and an evaluation of the quality of most commonly used miRNA prediction tools (TargetScan, PicTar, PITA, RNA22 and miRanda). To estimate efficiency of the miRNA–mRNA prediction tools, we used next parameters: sensitivity, positive predicted value, predictions in different mRNA regions (3' UTR, CDS, 5' UTR), predictions for different types of interactions (5 classes), predictions of “canonical” and “nocanonical” interactions, similarity with the random generated data. The analysis revealed low efficiency of all prediction programs in comparison with the CLASH data in terms of the all examined parameters.

BACKGROUND: Follicular lymphoma (FL) is an indolent malignancy of germinal center B-cell origin. FL patients experience remarkable heterogeneity in their disease trajectory, with many patients slowly progressing over several years, and a subset of patients experiencing an aggressive clinical course. Uncovering the cell-intrinsic and -extrinsic factors that govern differential progression and outcome in FL patients is thus essential. Beyond the genetic and epigenetic aberrations that contribute to FL oncogenesis, the tumor microenvironment (TME) plays an integral role in supporting the proliferation and survival of malignant cells. In a process described as "re-education", FL tumor cells may actively subvert the normal functions of non-malignant cells present in the TME, including T cells, follicular dendritic cells (FDCs), macrophages, dendritic cells, and stromal cells to support their survival and growth. METHODS: To address the importance of the TME in FL, we molecularly profiled excisional lymph node biopies from untreated FL patients using multiple platforms: bulk RNA sequencing (RNAseq), single-cell RNA sequencing (scRS), and a unique high content imaging method, Iterative Bleaching Extends MultipleXity (IBEX), which utilizes chemical bleaching to image 40+ proteins in the same tissue section by antibody staining. In combination with advanced computational tools for the quantitative analysis of cell types and distribution in tissues, we have used this approach to evaluate the TME:FL interaction within 8 FL samples, with 4 normal lymph nodes as controls. RESULTS: Both FL and TME components reconstructed from bulk RNA-seq were similar to the cellular composition revealed by scRS and IBEX analyses. Moreover, the bulk RNAseq and scRS identified the expression of genes involved in tumorigenesis and oncogenic signaling, often unique to each case. However, RNAseq-based approaches often miss important cellular and acellular components not readily extracted from dense tissues. For example, IBEX imaging can trace the pattern of blood vessels within a section, which cannot be achieved with non-imaging methods. In one case, our multi-parameter imaging studies revealed the close spatial interaction between clonal FL B cells, expressing a B-cell receptor (BCR) possessing a de novo N-linked glycosylation site introduced by somatic hypermutation, and cells expressing dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN). This contact may activate pro-survival signaling in the malignant B cells. CONCLUSIONS: Integration of bulk RNAseq, scRS, clonotype analysis, and IBEX reveals both shared and unique aspects of different FL tumors. These data highlight the importance of integrating direct tissue analysis by high-content imaging with methods examining aspects of isolated cells. This approach may provide a more complete understanding of tumor biology, which in turn will identify patients at risk for developing aggressive disease and rationally improve treatment strategies for FL. This research was supported in part by the Intramural Research Program of the NIH, NIAID and NCI Figure Disclosures Bagaev: BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Plotnikova:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Galkin:BostonGene: Current Employment, Patents & Royalties. Postovalova:BostonGene: Current Employment, Current equity holder in private company. Svekolkin:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Isaev:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Lozinsky:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Meerson:BostonGene: Current Employment. Varlamova:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Ovcharov:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Polyakova:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Nomie:BostonGene: Current Employment, Current equity holder in private company. Kotlov:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Tsiper:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Frenkel:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Attaulakhanov:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Fowler:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties.

Alanine-serine-cysteine transporter 2 (ASCT2, SLC1A5) is the primary transporter of glutamine in cancer cells and regulates the mTORC1 signaling pathway. The SLC1A5 function involves finely tuned orchestration of two domain movements that include the substrate-binding transport domain and the scaffold domain. Here, we present cryo-EM structures of human SLC1A5 and its complex with the substrate, L-glutamine in an outward-facing conformation. These structures reveal insights into the conformation of the critical ECL2a loop which connects the two domains, thus allowing rigid body movement of the transport domain throughout the transport cycle. Furthermore, the structures provide new insights into substrate recognition, which involves conformational changes in the HP2 loop. A putative cholesterol binding site was observed near the domain interface in the outward-facing state. Comparison with the previously determined inward-facing structure of SCL1A5 provides a basis for a more integrated understanding of substrate recognition and transport mechanism in the SLC1 family.

Alanine-serine-cysteine transporter 2 (ASCT2, SLC1A5) is the primary transporter of glutamine in cancer cells and regulates the mTORC1 signaling pathway. The SLC1A5 function involves finely tuned orchestration of two domain movements that include the substrate-binding transport domain and the scaffold domain. Here, we present cryo-EM structures of human SLC1A5 and its complex with the substrate, L-glutamine in an outward-facing conformation. These structures reveal insights into the conformation of the critical ECL2a loop which connects the two domains, thus allowing rigid body movement of the transport domain throughout the transport cycle. Furthermore, the structures provide new insights into substrate recognition, which involves conformational changes in the HP2 loop. A putative cholesterol binding site was observed near the domain interface in the outward-facing state. Comparison with the previously determined inward-facing structure of SCL1A5 provides a basis for a more integrated understanding of substrate recognition and transport mechanism in the SLC1 family. Our structures are likely to aid the development of potent and selective SLC1A5 inhibitors for the treatment of cancer and autoimmune disorders.

TTK/Mps1 is a key kinase controlling progression of cell division via participation in the mitotic spindle assembly checkpoint and is overexpressed in a number of human cancers. Herein we report the discovery of 4-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)benzamides as a potent, novel class of TTK inhibitors. The series was identified by means of bioisosteric replacement of the related imidazopyrazine and imidazopyridazine scaffolds. Optimization led to the identification of compounds with excellent potency (Ki=0.8nM) and exceptional kinase selectivity. The SAR indicates a strong dependence of activity on the presence of the N-cyclopropyl-2-methylbenzamide moiety delineating the geometry for 1½ type kinase inhibitor. Molecular modeling indicates the extensive and optimal contacts, mediated through H-bonds and hydrophobic interactions, are responsible for the selectivity and potency of the inhibitors. The compounds demonstrate a strong anti-proliferative activity in a panel of human cancer cell lines (HCT116 GI50

This work describes a scaffold hopping exercise that begins with known imidazo[1,2-a]pyrazines, briefly explores pyrazolo[1,5-a][1,3,5]triazines, and ultimately yields pyrazolo[1,5-a]pyrimidines as a novel class of potent TTK inhibitors. An X-ray structure of a representative compound is consistent with 1(1)/2 type inhibition and provides structural insight to aid subsequent optimization of in vitro activity and physicochemical and pharmacokinetic properties. Incorporation of polar moieties in the hydrophobic and solvent accessible regions modulates physicochemical properties while maintaining potency. Compounds with enhanced oral exposure were identified for xenograft studies. The work culminates in the identification of a potent (TTK K i = 0.1 nM), highly selective, orally bioavailable anticancer agent (CFI-402257) for IND enabling studies.

Fas is highly expressed on germinal center (GC) B cells, and mutations of FAS have been reported in diffuse large B-cell lymphoma (DLBCL); however, the function of Fas in the GC remains controversial. Although GC-derived DLBCL has better overall outcomes to therapy than other DLBCL types, some cases are refractory and the molecular basis for this is often unknown. We show that Fas is a strong cell-intrinsic regulator of GC B cells that promotes B-cell death in the light zone likely via T follicular helper (Tfh) cell-derived Fas ligand. In the absence of Fas, GCs were more clonally diverse due to an accumulation of cells that did not demonstrably bind antigen. We found that FAS alterations occurred most commonly in the GC-derived genetic subtype of DLBCL, EZB. FAS alterations in EZB were associated with inferior outcomes and an enrichment of Tfh cells. Alterations in FAS co-occurred with deficiency in HVEM and PD-1 ligands that regulate the Tfh-B cell interaction and were associated with increased diversity of B-cell receptor variable genes across samples. This work shows that Fas is critically required for GC homeostasis and suggests that loss of Tfh-mediated counterselection in the GC contributes to lethality in a distinct subtype of GC-derived lymphoma. Citation Format: Raud Razzaghi, Shreya Agarwal, Nikita Kotlov, Olga Plotnikova, Krystle Nomie, Da Wei Huang, George W. Wright, Moyi Li, Katsuyoshi Takata, Chen Yao, John J. O'Shea, James D. Phelan, Stefania Pittaluga, David W. Scott, Jagan R. Muppidi. Compromised counterselection by FAS creates a lethal subtype of germinal center lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-22.

Follicular lymphoma (FL) is the most common indolent non-Hodgkin's lymphoma; however, it remains incurable with conventional therapies and is poorly responsive to checkpoint blockade. Additionally, because FL develops so slowly (and often asymptomatically), a major research focus has been to avoid chemotherapy treatments to limit the potential development of treatment-related side effects and the risk of therapy-related second cancers. The mutational processes that lead to lymphomagenesis and progression also produce tumor-specific mutant antigens (TSMAs) that can be targeted by the immune system to control malignancies. Personalized cancer vaccines designed for these TSMAs represent a promising new strategy for treatment of FL. However, the feasibility of this approach and precisely how to optimize effective vaccine design, informed by next-generation sequencing data, are not fully understood. We hypothesize that (tumor/normal) whole-exome sequencing (WES) and (tumor) RNA sequencing (RNA-Seq) can be used to predict patient HLA typing and neoepitopes to engineer personalized cancer vaccines for FL. DNA and RNA from 58 patients' FL biopsies underwent WES and RNA-Seq. pVACtools and MiXCR predicted potential somatic and B-cell clonotype neoantigens, which were filtered to identify high-quality TSMAs. B-cell oligoclonality was determined by comparison to B-cell receptor (BCR) repertoire profiling of healthy individual lymph nodes. RNA-seq data allowed us to identify expressed TSMAs. Complementary in silico analysis based on mRNA-based peptide reconstruction and custom HLA affinity binding predictions were performed. An average of 52 somatic mutations per patient (range: 2-172) were identified. At least one high-quality TSMA was predicted for 57 of 58 patients. Five or more TSMA candidates were identified for 52 (90%) patients with a mean of 17 predicted peptides per patient (range: 0-45). 81% (813/1,004) of the total predicted TSMA peptides arose from missense mutations, 9% (94/1,004) from indels, and 10% (97/1,004) from BCR. 78% (45/58) of patients have both somatic and BCR vaccine candidates, while 21% (12/58) of patients had only somatic vaccine candidates. No fusion genes were identified within the cohort that could have been a source of neoepitope candidates. Predicted TSMAs were identified in multiple genes recurrently mutated in lymphoma (e.g., BCL2). There was a high prediction concordance with the orthogonal BostonGene Vaccine Module V1 pipeline. These preclinical results led to a first-in-human pilot trial of personalized TSMA vaccine combined with anti-PD-1 mAb for rel/ref FL patients (NCT03121677), with one response observed within 4 patients evaluable for response to date. TSMA peptides suitable for cancer vaccines were identified for most FL patients via next-generation sequencing, MiXCR and pVACtools. This preclinical study suggests that FL patients will be candidates for TSMA vaccine clinical trials, and pilot clinical results provide proof of concept for this approach. Citation Format: Cody A. Ramirez, Felix Frenkel, Olga Plotnikova, Vladislav Belousov, Alexander Bagaev, Elena Ocheredko, Susanna Kiwala, Jasreet Hundal, Zachary L. Skidmore, Marcus Watkins, Michelle Becker-Hapak, Thomas B. Mooney, Jason Walker, Catrina Fronick, Robert Fulton, Robert Schreiber, Nancy L. Bartlett, Brad Kahl, Ravshan Ataullakhanov, Malachi Griffith, Obi Griffith, Todd A. Fehniger. Identification of predicted neoantigen vaccine candidates in follicular lymphoma patients [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-56.

Gastric cancer (GC), responsible for ~700,000 deaths worldwide annually, is a dismal disease, with median survival for metastatic disease less than 1 year. We examined the efficacy and safety of cabazitaxel, a novel 3rd generation taxane, in the 2nd line treatment setting in advanced GC. Comprehensive correlative studies were performed to identify genetic aberrations or tumor microenvironment signatures associated with cabazitaxel activity. Patients with previously treated GC were eligible for this multicenter phase II study of single agent cabazitaxel (NCT01757171). The progression-free survival (PFS) rate at 3-mo using RECIST 1.1 was 28% (95%CI 17-42%) in taxane-naïve and 35% (95%CI 16-57%) in taxane treated cohorts. A fresh tissue biopsy of the tumor and matching adjacent non-tumor tissue was obtained from each of the 66 patients (87% of the study population) and was examined using whole exome sequencing (WES) and bulk RNAseq. We performed CIBERSORT deconvolution of the RNA expression data into its constituent immune cell types. Tumor samples were segregated into those with high or low macrophage M2 levels using the cohort specific median M2 abundance as the threshold. 26 tumor samples were examined for validation of the M2 signature by immunohistochemistry (IHC) using CD68 (pan-macrophage), CD163 (M2), and iNOS (M1) markers. GC WES showed numerous somatic alterations including missense mutations, chromosomal rearrangements, SNVs, small indels and CNAs with prevalent mutations in TP53 (26/47 cases), RHOA, and RTK/RAS signaling. Clinically actionable alterations included BRAF V600E, EGFR amplifications (10/47) and HER2 amplifications (8/47). One patient had a KRAS Q61H mutation predictive of resistance to a broad spectrum of RTK inhibitors. Other alterations included mutations in RTK signaling components, deletions of MTOR and STK11 suppressor gene and mutations in PI3K/mTOR pathway. We found that HER2 amplification was significantly more prevalent in responders, 50% HER2 positive among patients with PR/SD vs 10% in patients with PD (p=0.003). Patients with HER2 positive tumors had better PFS (p=0.04) and OS (45% 2-year survival vs 15%, p=0.002). Deconvolution analysis revealed an enrichment of an M2 macrophage signature in a cohort of patients having an improved PFS (45% vs 20% at 12 months, p=0.031). IHC analysis also showed M2 enrichment in 65% tissue samples examined (n=26). The M2-like macrophage signature was associated with improved outcome independent of HER2 amplification/ over-expression. In 8 out of 10 matched on-treatment biopsies, the M2-like signature significantly decreased post treatment. We have identified two novel biomarkers, HER2 overexpression and M2-high tumor macrophage signature, associated with improved outcomes in patients with GC treated with cabazitaxel. Additional correlative analyses and integration are underway. Citation Format: Sandipto Sarkar, Prashant K. Thakkar, Heinz Lenz, Peter Enzinger, Andrew H. Ko, Allyson J. Ocean, Yao Lu, Chao Zhang, Bhavneet Bhinder, Olga Plotnikova, Nikita Kotlov, Feliz Frenkel, Aleksander Bagaev, Olivier Elemento, Doron Betel, Paraskevi Giannakakou, Meredith E. Pittman, Manish A. Shah. HER2 expression and M2-like tumor infiltrating macrophages associated with Cabazitaxel activity in gastric cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2011.

8054 Background: Follicular lymphoma (FL) is incurable with conventional therapies and poorly responsive to immune checkpoint blockade. There is a need for new therapies without long-term complications of chemotherapy and with curative potential. We hypothesize that FL contains tumor-specific mutant antigens (TSMAs) that can be targeted by the immune system by vaccination. Recent reports have highlighted the potential for unique immunoglobulin peptides to elicit immune response in lymphomas. We utilized whole exome sequencing (WES) and RNA sequencing (RNA-Seq) of FL patient samples to infer HLA genotype, and predict TSMAs with the goal of designing a personalized cancer vaccine, supported by recent reports of this approach in solid cancers. Methods: DNA and RNA from 58 patients’ FL biopsies underwent WES and RNA-Seq. pVACtools and MiXCR predicted potential somatic and B-cell clonotype neoantigens, which were filtered to identify high quality TSMAs. B-cell oligoclonality was determined by comparison to B-cell receptor (BCR) repertoire profiling of healthy individual lymph nodes. RNA-seq data allowed us to identify expressed TSMAs. Complementary in silico analysis based on mRNA-based peptide reconstruction and custom HLA affinity binding predictions were performed. Results: An average of 52 somatic mutations per patient (range: 2-172) were identified. At least one high quality TSMA was predicted for 57 of 58 patients. Five or more TSMA candidates were identified for 52 (90%) patients with a mean of 17 predicted peptides per patient (range: 0-45). 81% (813/1,004) of the total predicted TSMA peptides arose from missense mutations, 9% (94/1,004) from indels, and 10% (97/1,004) from BCR. 78% (45/58) of patients have both somatic and BCR vaccine candidates, while 21% (12/58) of patients had only somatic vaccine candidates. Predicted TSMAs were identified in multiple genes recurrently mutated in lymphoma (e.g., BCL2). There was a high prediction concordance with the orthogonal BostonGene Vaccine Module V1 pipeline. These pre-clinical results led to a first-in-human pilot trial of personalized TSMA vaccine combined with anti-PD-1 mAb for rel/ref FL patients (NCT03121677), with one response observed within 4 patients evaluable for response to date. Conclusions: TSMA peptides suitable for cancer vaccines were identified for most FL patients via next-generation sequencing, MiXCR and pVACtools. This pre-clinical study suggests that FL patients will be candidates for TSMA vaccine clinical trials and pilot clinical results provide proof of concept for this approach.

The development of precision medicine approaches for diffuse large B cell lymphoma (DLBCL) is confounded by its pronounced genetic, phenotypic, and clinical heterogeneity. Recent multiplatform genomic studies revealed the existence of genetic subtypes of DLBCL using clustering methodologies. Here, we describe an algorithm that determines the probability that a patient's lymphoma belongs to one of seven genetic subtypes based on its genetic features. This classification reveals genetic similarities between these DLBCL subtypes and various indolent and extranodal lymphoma types, suggesting a shared pathogenesis. These genetic subtypes also have distinct gene expression profiles, immune microenvironments, and outcomes following immunochemotherapy. Functional analysis of genetic subtype models highlights distinct vulnerabilities to targeted therapy, supporting the use of this classification in precision medicine trials.

Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.

TTK kinase was identified by in-house siRNA screen and pursued as a tractable, novel target for cancer treatment. A screening campaign and systematic optimization, supported by computer modeling led to an indazole core with key sulfamoylphenyl and acetamido moieties at positions 3 and 5, respectively, establishing a novel chemical class culminating in identification of 72 (CFI-400936). This potent inhibitor of TTK (IC50 = 3.6 nM) demonstrated good activity in cell based assay and selectivity against a panel of human kinases. A co-complex TTK X-ray crystal structure and results of a xenograft study with TTK inhibitors from this class are described.

Previous publications from our laboratory have introduced novel inhibitors of Polo-like kinase 4 (PLK4), a mitotic kinase identified as a potential target for cancer therapy. The search for potent and selective PLK4 inhibitors yielded (E)-3-((1H-indazol-6-yl)methylene)indolin-2-ones, which were superseded by the bioisosteric 2-(1H-indazol-6-yl)spiro[cyclopropane-1,3′-indolin]-2′-ones, e.g., 3. The later scaffold confers improved drug-like properties and incorporates two stereogenic centers. This work reports the discovery of a novel one-pot double SN2 displacement reaction for the stereoselective installation of the desired asymmetric centers and confirms the stereochemistry of the most potent stereoisomer, e.g., 44. Subsequent work keys on the optimization of the oral exposure of nanomolar PLK4 inhibitors with potent cancer cell growth inhibitory activity. A short list of compounds with superior potency and pharmacokinetic properties in rodents and dogs was studied in mouse models of tumor growth. We con...

The discovery, structure-based design, synthesis, and optimization of NIK inhibitors are described. Our work began with an HTS hit, imidazopyridinyl pyrimidinamine 1. We utilized homology modeling and conformational analysis to optimize the indole scaffold leading to the discovery of novel and potent conformationally constrained inhibitors such as compounds 25 and 28. Compounds 25 and 31 were co-crystallized with NIK kinase domain to provide structural insights.

Background: NIK is a central component in the non-canonical NF-κB pathway, and its activity is associated with various diseases. Results: An N-terminal extension is required for activity and stabilizes the kinase in an active conformation. Conclusion: The NIK kinase domain adopts a constitutively active conformation. Significance: This work presents the first NIK structure and provides a molecular basis for NIK regulation., NF-κB-inducing kinase (NIK) is a central component in the non-canonical NF-κB signaling pathway. Excessive NIK activity is implicated in various disorders, such as autoimmune conditions and cancers. Here, we report the first crystal structure of truncated human NIK in complex with adenosine 5′-O-(thiotriphosphate) at a resolution of 2.5 Å. This truncated protein is a catalytically active construct, including an N-terminal extension of 60 residues prior to the kinase domain, the kinase domain, and 20 residues afterward. The structure reveals that the NIK kinase domain assumes an active conformation in the absence of any phosphorylation. Analysis of the structure uncovers a unique role for the N-terminal extension sequence, which stabilizes helix αC in the active orientation and keeps the kinase domain in the catalytically competent conformation. Our findings shed light on the long-standing debate over whether NIK is a constitutively active kinase. They also provide a molecular basis for the recent observation of gain-of-function activity for an N-terminal deletion mutant (ΔN324) of NIK, leading to constitutive non-canonical NF-κB signaling with enhanced B-cell adhesion and apoptosis resistance.

The nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARgamma) plays central roles in adipogenesis and glucose homeostasis and is the molecular target for the thiazolidinedione (TZD) class of antidiabetic drugs. Activation of PPARgamma by TZDs improves insulin sensitivity; however, this is accompanied by the induction of several undesirable side effects. We have identified a novel synthetic PPARgamma ligand, T2384, to explore the biological activities associated with occupying different regions of the receptor ligand-binding pocket. X-ray crystallography studies revealed that T2384 can adopt two distinct binding modes, which we have termed "U" and "S", interacting with the ligand-binding pocket of PPARgamma primarily via hydrophobic contacts that are distinct from full agonists. The different binding modes occupied by T2384 induced distinct patterns of coregulatory protein interaction with PPARgamma in vitro and displayed unique receptor function in cell-based activity assays. We speculate that these unique biochemical and cellular activities may be responsible for the novel in vivo profile observed in animals treated systemically with T2384. When administered to diabetic KKAy mice, T2384 rapidly improved insulin sensitivity in the absence of weight gain, hemodilution, and anemia characteristics of treatment with rosiglitazone (a TZD). Moreover, upon coadministration with rosiglitazone, T2384 was able to antagonize the side effects induced by rosiglitazone treatment alone while retaining robust effects on glucose disposal. These results are consistent with the hypothesis that interactions between ligands and specific regions of the receptor ligand-binding pocket might selectively trigger a subset of receptor-mediated biological responses leading to the improvement of insulin sensitivity, without eliciting less desirable responses associated with full activation of the receptor. We suggest that T2384 may represent a prototype for a novel class of PPARgamma ligand and, furthermore, that molecules sharing some of these properties would be useful for treatment of type 2 diabetes.

The acetamido and carboxamido substituted 3-(1H-indazol-3-yl)benzenesulfonamides are potent TTK inhibitors. However, they display modest ability to attenuate cancer cell growth; their physicochemical properties, and attendant pharmacokinetic parameters, are not drug-like. By eliminating the polar 3-sulfonamide group and grafting a heterocycle at the 4 position of the phenyl ring, potent inhibitors with oral exposure were obtained. An X-ray cocrystal structure and a refined binding model allowed for a structure guided approach. Systematic optimization resulted in novel TTK inhibitors, namely 3-(4-(heterocyclyl)phenyl)-1H-indazole-5-carboxamides. Compounds incorporating the 3-hydroxy-8-azabicyclo[3.2.1]octan-8-yl bicyclic system were potent (TTK IC5010 nM, HCT116 GI500.1 μM), displayed low off-target activity (500×), and microsomal stability (T(1/2)30 min). A subset was tested in rodent PK and mouse xenograft models of human cancer. Compound 75 (CFI-401870) recapitulated the phenotype of TTK RNAi, demonstrated in vivo tumor growth inhibition upon oral dosing, and was selected for preclinical evaluation.

Previous publications from our laboratory have introduced novel inhibitors of Polo-like kinase 4 (PLK4), a mitotic kinase identified as a potential target for cancer therapy. The search for potent and selective PLK4 inhibitors yielded (E)-3-((1Hindazol-6-yl)methylene)indolin-2-ones, which were superseded by the bioisosteric 2-(1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-ones, e.g., 3. The later scaffold confers improved drug-like properties and incorporates two stereogenic centers. This work reports the discovery of a novel one-pot double SN2 displacement reaction for the stereoselective installation of the desired asymmetric centers and confirms the stereochemistry of the most potent stereoisomer, e.g., 44. Subsequent work keys on the optimization of the oral exposure of nanomolar PLK4 inhibitors with potent cancer cell growth inhibitory activity. A short list of compounds with superior potency and pharmacokinetic properties in rodents and dogs was studied in mouse models of tumor growth. We conclude with the identification of compound 48 (designated CFI-400945) as a novel clinical candidate for cancer therapy.

Lysine acetylation of the tumor suppressor protein p53 in response to a wide variety of cellular stress signals is required for its activation as a transcription factor that regulates cell cycle arrest, senescence, or apoptosis. Here, we report that the conserved bromo-domain of the transcriptional coactivator CBP (CREB binding protein) binds specifically to p53 at the C-terminal acetylated lysine 382. This bromodomain/acetyl-lysine binding is responsible for p53 acetylation-dependent coactivator recruitment after DNA damage, a step essential for p53-induced transcriptional activation of the cyclin-dependent kinase inhibitor p21 in G1 cell cycle arrest. We further present the three-dimensional nuclear magnetic resonance structure of the CBP bromodomain in complex with a lysine 382-acetylated p53 peptide. Using structural and biochemical analyses, we define the molecular determinants for the specificity of this molecular recognition.

Inactivation of mitogen-activated protein kinases (MAPKs) by MAPK phosphatases (MKPs) is accomplished via substrate-induced activation of the latter enzymes; however, the structural basis for the underlying mechanism remains elusive. Here, we report the three-dimensional solution structure of the C-terminal phosphatase domain of the prototypical MKP PAC-1, determined when bound to phosphate. Structural and biochemical analyses reveal unique active site geometry of the enzyme important for binding to phosphorylated threonine and tyrosine of MAPK ERK2. Our study further demonstrates that the dynamic interaction between the N-terminal kinase binding domain and the C-terminal phosphatase domain of an MKP is directly coupled to MAPK-induced conformational change of the phosphatase active site, which is essential for eliciting its full enzymatic activity.

Polo-like kinase 4 (PLK4), a unique member of the polo-like kinase family of serine-threonine kinases, is a master regulator of centriole duplication that is important for maintaining genome integrity. Overexpression of PLK4 is found in several human cancers and is linked with a predisposition to tumorigenesis. Previous efforts to identify potent and efficacious PLK4 inhibitors resulted in the discovery of (E)-3-((1H-indazol-6-yl)methylene)indolin-2-ones, which are superseded by the bioisosteric 2-(1H-indazol-6-yl)spiro[cyclopropane-1,3′-indolin]-2′-ones reported herein. Optimization of this new cyclopropane-linked series was based on a computational model of a PLK4 X-ray structure and SAR attained from the analogous alkenelinked series. The racemic cyclopropane-linked compounds showed PLK4 affinity and antiproliferative activity comparable to their alkene-linked congeners with improved hysicochemical, ADME, and pharmacokinetic properties. Positive xenograft results from the MDA-MB-468 human breast cancer xenograft model for compound 18 support the investigation of PLK4 inhibitors as anticancer therapeutics. A PLK4 X-ray co-structure with racemate 18 revealed preferential binding of the 1R,2S enantiomer to the PLK4 kinase domain.

MAP kinases (MAPKs), which control mitogenic signal transduction in all eukaryotic organisms, are inactivated by dual specificity MAPK phosphatases (MKPs). MKP-3, a prototypical MKP, achieves substrate specificity through its N-terminal domain binding to the MAPK ERK2, resulting in the activation of its C-terminal phosphatase domain. The solution structure and biochemical analysis of the ERK2 binding (EB) domain of MKP-3 show that regions that are essential for ERK2 binding partly overlap with its sites that interact with the C-terminal catalytic domain, and that these interactions are functionally coupled to the active site residues of MKP-3. Our findings suggest a novel mechanism by which the EB domain binding to ERK2 is transduced to cause a conformational change of the C-terminal catalytic domain, resulting in the enzymatic activation of MKP-3.

SNT adaptor proteins transduce activation of fibroblast growth factor receptors (FGFRs) and neurotrophin receptors (TRKs) to common signaling targets. The SNT-1 phosphotyrosine binding (PTB) domain recognizes activated TRKs at a canonical NPXpY motif and, atypically, binds to nonphosphorylated FGFRs in a region lacking tyrosine or asparagine. Here, using NMR and mutational analyses, we show that the PTB domain utilizes distinct sets of amino acid residues to interact with FGFRs or TRKs in a mutually exclusive manner. The FGFR1 peptide wraps around the β sandwich structure of the PTB domain, and its binding is possibly regulated by conformational change of a unique C-terminal β strand in the protein. Our results suggest mechanisms by which SNTs serve as molecular switches to mediate the essential interplay between FGFR and TRK signaling during neuronal differentiation.

The family of Polo-like kinases is important in the regulation of mitotic progression; this work keys on one member, namely Polo-like kinase 4 (PLK4). PLK4 has been identified as a candidate anticancer target which prompted a search for potent and selective inhibitors of PLK4. The body of the paper describes lead generation and optimization work which yielded nanomolar PLK4 inhibitors. Lead generation began with directed virtual screening, using a ligand-based focused library and a PLK4 homology model. Validated hits were used as starting points for the design and discovery of PLK4 inhibitors of novel structure, namely (E)-3-((1H-indazol-6-yl)methylene)indolin-2-ones. Computational models, based on a published X-ray structure (PLK4 kinase domain), were used to understand and optimize the in vitro activity of the series; potent antiproliferative activity was obtained. The kinase selectivity profile and cell cycle analysis of selected inhibitors are described. The results of a xenograft study with an optimized compound 50 (designated CFI-400437) support the potential of these novel PLK4 inhibitors for cancer therapy.

The tandem PHD finger-bromodomain, found in many chromatin-associated proteins, has an important role in gene silencing by the human co-repressor KRAB-associated protein 1 (KAP1). Here we report the three-dimensional solution structure of the tandem PHD finger-bromodomain of KAP1. The structure reveals a distinct scaffold unifying the two protein modules, in which the first helix, alpha(Z), of an atypical bromodomain forms the central hydrophobic core that anchors the other three helices of the bromodomain on one side and the zinc binding PHD finger on the other. A comprehensive mutation-based structure-function analysis correlating transcriptional repression, ubiquitin-conjugating enzyme 9 (UBC9) binding and SUMOylation shows that the PHD finger and the bromodomain of KAP1 cooperate as one functional unit to facilitate lysine SUMOylation, which is required for KAP1 co-repressor activity in gene silencing. These results demonstrate a previously unknown unified function for the tandem PHD finger-bromodomain as an intramolecular small ubiquitin-like modifier (SUMO) E3 ligase for transcriptional silencing.

Inactivation of mitogen-activated protein kinases (MAPKs) by MAPK phosphatases (MKPs) is accomplished via substrate-induced activation of the latter enzymes; however, the structural basis for the underlying mechanism remains elusive. Here, we report the three-dimensional solution structure of the C-terminal phosphatase domain of the prototypical MKP PAC-1, determined when bound to phosphate. Structural and biochemical analyses reveal unique active site geometry of the enzyme important for binding to phosphorylated threonine and tyrosine of MAPK ERK2. Our study further demonstrates that the dynamic interaction between the N-terminal kinase binding domain and the C-terminal phosphatase domain of an MKP is directly coupled to MAPK-induced conformational change of the phosphatase active site, which is essential for eliciting its full enzymatic activity.

Phosphotyrosine binding (PTB) domains of the adaptor protein Shc and insulin receptor substrate (IRS-1) interact with a distinct set of activated and tyrosine-phosphorylated cytokine and growth factor receptors and play important roles in mediating mitogenic signal transduction. By using the technique of isothermal titration calorimetry, we have studied the thermodynamics of binding of the Shc and IRS-1 PTB domains to tyrosine-phosphorylated NPXY-containing peptides derived from known receptor binding sites. The results showed that relative contributions of enthalpy and entropy to the free energy of binding are dependent on specific phosphopeptides. Binding of the Shc PTB domain to tyrosine-phosphorylated peptides from TrkA, epidermal growth factor, ErbB3, and insulin receptors is achieved via an overall entropy-driven reaction. On the other hand, recognition of the phosphopeptides of insulin and interleukin-4 receptors by the IRS-1 PTB domain is predominantly an enthalpy-driven process. Mutagenesis and amino acid substitution experiments showed that in addition to the tyrosine-phosphorylated NPXY motif, the PTB domains of Shc and IRS-1 prefer a large hydrophobic residue at pY-5 and a small hydrophobic residue at pY-1, respectively (where pY is phosphotyrosine). These results agree with the calculated solvent accessibility of these two key peptide residues in the PTB domain/peptide structures and support the notion that the PTB domains of Shc and IRS-1 employ functionally distinct mechanisms to recognize tyrosine-phosphorylated receptors.