Boehringer Ingelheim (Austria)

Understanding similarities between protein binding sites has long been of great interest, as such comparisons can reveal functional relationships that transcend sequence or fold. However, systematic comparison remains challenging due to the difficulty of defining active sites consistently and developing descriptors that are both general and discriminative. We present binding site vectors, a computational framework for a high-resolution comparison of macromolecular binding sites that integrates both structural and electrostatic properties. The vectors extend spherically from the center of the pocket, terminating at its surface to capture shape and electrostatic features in a multidimensional manner. Geometrically anchored, they enable a systematic comparison of binding sites across diverse systems. We applied this approach to cytochrome P450 (CYP) enzymes, analyzing over 600 human and plant CYP structures and a subset of 23 extensive structural ensembles obtained through molecular dynamics (MD) simulation. Comparisons based on binding site vectors reveal structural-functional relationships missed by sequence- or backbone-based groupings, particularly when full conformational ensembles are included. This demonstrates that binding site vectors provide a robust framework for both functional classification and deep mechanistic insights into macromolecular systems.

KRAS is among the most frequently mutated oncogenes in cancer, and for decades, efforts at pharmacological blockade of its function in solid cancers have been unsuccessful. A notable advance in this endeavor is the recent development of small-molecule KRAS inhibitors, which enable direct targeting of the mutant oncoprotein. Here, we comprehensively evaluated the preclinical efficacy of BI-2493, a first-in-class allele-agnostic mutant-KRAS inhibitor (panKRASi), in pancreatic ductal adenocarcinoma (PDAC). We report effective tumor growth suppression across a broad range of models, including cell lines, patient-derived xenografts (PDXs), and syngeneic orthotopic models, and prolonged survival in genetically engineered mouse models. Overall, transcriptomic, proteomic, and phosphoproteomic profiling of panKRASi-treated models confirmed RAS pathway inhibition along with up-regulation of LKB1/AMPK (liver kinase B1/AMP-activated protein kinase) targets. In panKRASi-treated immune-replete models, we observed increased intratumoral CD8+ effector T cells and decreased infiltration of myeloid cells, along with remodeling of the tumor microenvironment (TME), enabling responses to immune checkpoint blockade. In the long term, emergence of resistance to panKRASi monotherapy was associated with increased YAP signaling within tumor cells and enhanced expression of immune checkpoints in the TME that impede effective T cell function. Our multifaceted approach identified potential combinatorial approaches for generating sustained responses to panKRASi.

BackgroundFor many solid types of cancer including colorectal cancer treatment with the VEGF antibody bevacizumab as anti-angiogenic treatment has become standard of care. Nevertheless, long-term treatment with anti-angiogenic drugs in combination with other treatment modalities or alone can induce resistance through alternative pro-angiogenic pathways. In this study, we investigated the effects of nintedanib, an anti- VEGFR/PDGFR/FGFR kinase inhibitor, on tumor vasculature to determine the potential benefits of combined inhibition of multiple pro-angiogenic factors.MethodsIn a colorectal xenograft model, subcutaneous tumors were treated with Nintedanib. Tumor growth patterns were measured and tumors were analysed histologically regarding effects on tumor angiogenesis and parameters of vascular normalization.ResultsInhibition of VEGFR/PDGFR/FGFR by Nintedanib was able to reduce tumor growth by significantly inhibiting angiogenesis and inducing tumor cell death. The remaining vessels showed decreased vascular leakage and improved oxygen delivery, indicating a functionally and structurally improved vascular bed resulting from vascular normalization.ConclusionIn xenograft mouse model of colorectal cancer Nintedanib revealed anti-tumoral effects and induced vascular normalization. Our findings indicate that the treatment with Nintedanib could be able to improve intratumoral oxygen and thereby drug delivery to potentially enhance the efficacy of preexisting oncological therapies such as chemotherapy and radiation.

Oncogenic gene fusions occur across a broad range of cancers and are a defining feature of some cancer types. Cancers driven by gene fusion products tend to respond well to targeted therapies, where available; thus, detection of potentially targetable oncogenic fusions is necessary to select optimal treatment. Detection methods include non-sequencing methods, such as fluorescence in situ hybridization and immunohistochemistry, and sequencing methods, such as DNA- and RNA-based next-generation sequencing (NGS). While NGS is an efficient way to analyze multiple genes of interest at once, economic and technical factors may preclude its use in routine care globally, despite several guideline recommendations. The aim of this review is to present a summary of oncogenic gene fusions, with a focus on fusions that affect tyrosine kinase signaling, and to highlight the importance of testing for oncogenic fusions. We present an overview of the identification of oncogenic gene fusions and therapies approved for the treatment of cancers harboring gene fusions, and summarize data regarding treating fusion-positive cancers with no current targeted therapies and clinical studies of fusion-positive cancers. Although treatment options may be limited for patients with rare alterations, healthcare professionals should identify patients most likely to benefit from oncogenic gene fusion testing and initiate the appropriate targeted therapy to achieve optimal treatment outcomes.

We evaluated the in vivo therapeutic efficacy and tolerability of BI-3406-mediated pharmacological inhibition of SOS1 in comparison to genetic ablation of this universal Ras-GEF in various KRAS-dependent experimental tumor settings. Contrary to the rapid lethality caused by SOS1 genetic ablation in SOS2KO mice, SOS1 pharmacological inhibition by its specific inhibitor BI-3406 did not significantly affect animal weight/viability nor cause noteworthy systemic toxicity. Allograft assays using different KRASmut cell lines showed that treatment with BI-3406 impaired RAS activation and RAS downstream signaling and decreased tumor burden and disease progression as a result of both tumor-intrinsic and -extrinsic therapeutic effects of the drug. Consistent with prior genetic evidence and the KRASmut allografts assays in immunocompromised mice, our analyses using an in vivo model of KRASG12D-driven lung adenocarcinoma (LUAD) in immunocompetent mice showed that single, systemic BI-3406 treatment impaired tumor growth and downmodulated protumorigenic components of the tumor microenvironment comparably to SOS1 genetic ablation or to treatment with the specific KRASG12D inhibitor MRTX1133. Furthermore, markedly stronger, synergistic antitumor effects were observed upon concomitant treatment with BI-3406 and MRTX1133 in the same in vivo LUAD mouse model. Our data confirm SOS1 as an actionable therapy target in RAS-dependent cancers and suggest that BI-3406 treatment may yield clinical benefit both as monotherapy or as a potential combination partner for multiple RAS-targeting strategies.

In this study, we developed a customized high-resolution mass spectrometry metabolomics workflow integrating the dual sugar test employing lactulose and mannitol as test probes for intestinal permeability assessment with untargeted screening of small molecules. Urine samples were collected from patients with major depression and healthy controls as part of a clinical study at the psychiatric department. Using a dual injection/dual chromatography setup, the test probes were quantified by hydrophilic interaction liquid chromatography (HILIC) in a targeted assay, while drugs and their metabolites were profiled in an untargeted manner by reversed-phase separation. Rigorous method development and validation allowed for selective separation of sugar isomers and consequently accurate quantification of lactulose and mannitol in urine. Internal standardization with compound specific stable isotope-labeled standards enabled excellent analytical figures of merit such as high recoveries, precision (< 5%), and working range (5 orders of magnitude). Within one analytical run, intestinal permeability was assessed together with drugs and their metabolites, allowing to screen for confounding drugs and patient compliance to the therapeutic scheme.Graphical

ObjectiveA phase 2 trial tested different doses of the anti‐CD40 monoclonal antibody BI 655064 as an add‐on therapy to the standard of care in patients with class III or IV lupus nephritis (LN) with active disease. A post hoc analysis showed a potential benefit of the higher tested doses (180 and 240 mg) versus a low dose (120 mg) or placebo. We investigated whether the treatment effect of BI 655064 on kidney outcomes may be modified by the presence of glomerular monocytes, a target for this drug with a well‐known role in LN pathogenesis.MethodsOne hundred one renal biopsies of patients with LN enrolled in the BI 655064 trial were scored centrally. The estimated glomerular filtration rate (eGFR), spot urine protein/urine creatinine ratio (UP/UC), and complete renal response (CRR) were evaluated over 52 weeks. Patients were divided according to a “better” or “worse” performance than the average of all patients in the cohort, predicted by a mixed model for repeated measurements. Logistic regression models adjusted for potential confounders were used to assess the association between different treatment doses and outcomes according to the presence or absence of monocytes.ResultsA higher BI 655064 dose (180 or 240 mg) was associated with better outcomes of UP/UC and CRR when glomerular monocytes were present in kidney biopsy samples (odds ratio [OR] 3.66 [95% confidence interval (CI) 1.09–12.3], P = 0.04; OR 4.58 [95% CI 1.24–16.9], P = 0.02). A trend toward improved eGFR was also observed in these patients (at 52 weeks, P = 0.08).ConclusionIn LN kidney biopsy samples with glomerular monocytes, high‐dose BI 655064 treatment improved proteinuria at 52 weeks and resulted in a higher CRR compared to biopsy samples without glomerular monocytes. Histologic features may guide the choice of treatment for individual patients with LN.

Drug discovery in oncology is characterized by high attrition rates in clinical trials. The main reasons are lack of efficacy or unacceptable toxicity. Thus, more predictive preclinical models for drug discovery and development are urgently needed. In the field of immune-oncology, preclinical models are particularly demanding since they have to reflect the complex interplay between tumor cells and immune cells in the human body.The recent years were characterized by vast advancement in 3D in vitro models and organoid techniques. These models mimic the tumor microenvironment more realistically than traditional 2D cell cultures. They can include the main components of the tumor microenvironment, like (primary) tumor cells, immune cells and other stromal cells and allow growth and interaction of the diverse cell types in 3D. Thus, these 3D models provide a physiologically relevant platform for studying the mode of action of (immuno-) oncologic therapeutic drugs. SMAC (second mitochondrial-derived activator of caspases) mimetics, like many other anticancer drugs, can exert their effects on different cell populations within the tumor microenvironment, which can be analyzed by using 3D co-cultures as preclinical models.Fluorescence microscopy is a powerful tool to study biological processes. In live cell imaging, entire cells or subcellular structures can be monitored over time with the help of fluorescent labels.In this chapter, we describe a human 3D co-culture infiltration assay combining tumor cells embedded in a hydrogel and immune cells added on top of the hydrogel. This 3D co-culture is stable for more than 1week and gives insights into cellular drug responses over time. We used live cell imaging/fluorescence microscopy as the main readout to quantify immune cell infiltration in 3D in response to SMAC mimetic treatment.