Drug-microenvironment perturbations reveal resistance mechanisms and prognostic subgroups in CLL.

The tumour microenvironment and genetic alterations collectively influence drug efficacy in cancer, but current evidence is limited and systematic analyses are lacking. Using chronic lymphocytic leukaemia (CLL) as a model disease, we investigated the influence of 17 microenvironmental stimuli on 12 drugs in 192 genetically characterised patient samples. Based on microenvironmental response, we identified four subgroups with distinct clinical outcomes beyond known prognostic markers. Response to multiple microenvironmental stimuli was amplified in trisomy 12 samples. Trisomy 12 was associated with a distinct epigenetic signature. Bromodomain inhibition reversed this epigenetic profile and could be used to target microenvironmental signalling in trisomy 12 CLL. We quantified the impact of microenvironmental stimuli on drug response and their dependence on genetic alterations, identifying interleukin 4 (IL4) and Toll-like receptor (TLR) stimulation as the strongest actuators of drug resistance. IL4 and TLR signalling activity was increased in CLL-infiltrated lymph nodes compared with healthy samples. High IL4 activity correlated with faster disease progression. The publicly available dataset can facilitate the investigation of cell-extrinsic mechanisms of drug resistance and disease progression.

Genetic characterization of B-cell prolymphocytic leukemia: a prognostic model involving MYC and TP53.

B-cell prolymphocytic leukemia (B-PLL) is a rare hematological disorder whose underlying oncogenic mechanisms are poorly understood. Our cytogenetic and molecular assessments of 34 patients with B-PLL revealed several disease-specific features and potential therapeutic targets. The karyotype was complex (≥3 abnormalities) in 73% of the patients and highly complex (≥5 abnormalities) in 45%. The most frequent chromosomal aberrations were translocations involving MYC [t(MYC)] (62%), deletion (del)17p (38%), trisomy (tri)18 (30%), del13q (29%), tri3 (24%), tri12 (24%), and del8p (23%). Twenty-six (76%) of the 34 patients exhibited an MYC aberration, resulting from mutually exclusive translocations or gains. Whole-exome sequencing revealed frequent mutations in TP53, MYD88, BCOR, MYC, SF3B1, SETD2, CHD2, CXCR4, and BCLAF1. The majority of B-PLL used the IGHV3 or IGHV4 subgroups (89%) and displayed significantly mutated IGHV genes (79%). We identified 3 distinct cytogenetic risk groups: low risk (no MYC aberration), intermediate risk (MYC aberration but no del17p), and high risk (MYC aberration and del17p) (P = .0006). In vitro drug response profiling revealed that the combination of a B-cell receptor or BCL2 inhibitor with OTX015 (a bromodomain and extra-terminal motif inhibitor targeting MYC) was associated with significantly lower viability of B-PLL cells harboring a t(MYC). We concluded that cytogenetic analysis is a useful diagnostic and prognostic tool in B-PLL. Targeting MYC may be a useful treatment option in this disease.

TRRAP is essential for regulating the accumulation of mutant and wild-type p53 in lymphoma.

Tumors accumulate high levels of mutant p53 (mutp53), which contributes to mutp53 gain-of-function properties. The mechanisms that underlie such excessive accumulation are not fully understood. To discover regulators of mutp53 protein accumulation, we performed a large-scale RNA interference screen in a Burkitt lymphoma cell line model. We identified transformation/transcription domain-associated protein (TRRAP), a constituent of several histone acetyltransferase complexes, as a critical positive regulator of both mutp53 and wild-type p53 levels. TRRAP silencing attenuated p53 accumulation in lymphoma and colon cancer models, whereas TRRAP overexpression increased mutp53 levels, suggesting a role for TRRAP across cancer entities and p53 mutations. Through clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screening, we identified a 109-amino-acid region in the N-terminal HEAT repeat region of TRRAP that was crucial for mutp53 stabilization and cell proliferation. Mass spectrometric analysis of the mutp53 interactome indicated that TRRAP silencing caused degradation of mutp53 via the MDM2-proteasome axis. This suggests that TRRAP is vital for maintaining mutp53 levels by shielding it against the natural p53 degradation machinery. To identify drugs that alleviated p53 accumulation similarly to TRRAP silencing, we performed a small-molecule drug screen and found that inhibition of histone deacetylases (HDACs), specifically HDAC1/2/3, decreased p53 levels to a comparable extent. In summary, here we identify TRRAP as a key regulator of p53 levels and link acetylation-modifying complexes to p53 protein stability. Our findings may provide clues for therapeutic targeting of mutp53 in lymphoma and other cancers.

MED12 mutations and NOTCH signalling in chronic lymphocytic leukaemia.

Mutations in the N-terminus of MED12 protein occur at high frequency in uterine leiomyomas and breast fibroepithelial tumours, and are frequently found in chronic lymphocytic leukaemia (CLL). MED12 mutations have been previously linked to aberrant Cyclin C-CDK8 kinase activity, but the exact oncogenic function in CLL is unknown. Here, we characterized MED12 mutations in CLL and identified recurrent mutations in 13 out of 188 CLL patients (6·9%), which clustered in the N-terminus. MED12 mutations were associated with unmutated IGHV (P = 0·024). Protein analysis of NOTCH1 in primary CLL samples revealed increased levels of NOTCH1 intracellular domain (NICD), the active form of NOTCH1, in the context of MED12 mutations. We found evidence that NICD is the target of Cyclin C-CDK8 kinase using a specific CDK8 inhibitor. In line with these findings, MED12 mutations were mutually exclusive to mutations in NOTCH1 in CLL, based on a meta-analysis of 1429 CLL patients (P = 0·011). Our results suggest that MED12 mutations may contribute to CLL pathogenesis by activating NOTCH signalling.

Perioperative Enhanced Recovery Concepts Significantly Improve Postoperative Outcome in Patients with Crohn`s Disease.

BACKGROUND AND AIMS: Despite recent advancements in medical and surgical techniques in patients suffering from Crohn`s Disease (CD), postoperative morbidity remains relevant due to a long-standing, non-curable disease burden. As demonstrated for oncological patients, perioperative enhanced recovery concepts provide great potential to improve postoperative outcome. However, robust evidence about the effect of perioperative enhanced recovery concepts in the specific cohort of CD patients is lacking. METHODS: In a prospective single-center study, all patients receiving ileocecal resection due to CD between 2020 and 2023 were included. A specific perioperative enhanced recovery concept (ERC) was implemented and patients were divided into two groups (before and after implementation). The primary outcome focused on postoperative complications as measured by the Comprehensive Complication Index (CCI), secondary endpoints were severe complications, length of hospital stay, and rates of re-admission. RESULTS: 83 patients were analyzed of which 33 patients participated in the enhanced recovery program (postERC). While patient characteristics were comparable between both groups, ERC resulted in significantly decreased rates of overall and severe postoperative complications (CCI: 21.4 versus 8.4, p=0.0036; Clavien Dindo >2: 38% versus 3.1%, p=0.0002). Additionally, postERC-patients were earlier ready for discharge (6.5 days versus 5 days, p=0.001) and rates of re-admission were significantly lower (20% versus 3.1%, p=0.03). In a multivariate analysis, the recovery concept was identified as independent factor to reduce severe postoperative complications (p=0.019). CONCLUSION: A specific perioperative enhanced recovery concept significantly improves the postoperative outcome of patients suffering from Crohn`s Disease.

Broad-spectrum antimalarial activity of peptido sulfonyl fluorides, a new class of proteasome inhibitors.

Despite declining numbers of cases and deaths, malaria remains a major public health problem in many parts of the world. Today, case management relies heavily on a single class of antimalarial compounds: artemisinins. Hence, development of resistance against artemisinins may destroy current malaria control strategies. Beyond malaria control are elimination and eradication programs that will require drugs with good activity against acute infection but also with preventive and transmission-blocking properties. Consequently, new antimalarials are needed not only to ensure malaria control but also for elimination and eradication efforts. In this study, we introduce peptido sulfonyl fluorides (PSF) as a new class of compounds with antiplasmodial activity. We show that PSF target the plasmodial proteasome and act on all asexual stages of the intraerythrocytic cycle and on gametocytes. PSF showed activities at concentrations as low as 20 nM against multidrug-resistant and chloroquine-sensitive Plasmodium falciparum laboratory strains and clinical isolates from Gabon. Structural requirements for activity were identified, and cytotoxicity in human HeLa or HEK 293 cells was low. The lead PSF PW28 suppressed growth of Plasmodium berghei in vivo but showed signs of toxicity in mice. Considering their modular structure and broad spectrum of activity against different stages of the plasmodial life cycle, proteasome inhibitors based on PSF have a great potential for further development as preclinical candidate compounds with improved species-specific activity and less toxicity.

Aberrant gene activation in synovial sarcoma relies on SSX specificity and increased PRC1.1 stability.

The SS18-SSX fusion drives oncogenic transformation in synovial sarcoma by bridging SS18, a member of the mSWI/SNF (BAF) complex, to Polycomb repressive complex 1 (PRC1) target genes. Here we show that the ability of SS18-SSX to occupy H2AK119ub1-rich regions is an intrinsic property of its SSX C terminus, which can be exploited by fusion to transcriptional regulators beyond SS18. Accordingly, SS18-SSX recruitment occurs in a manner that is independent of the core components and catalytic activity of BAF. Alternative SSX fusions are also recruited to H2AK119ub1-rich chromatin and reproduce the expression signatures of SS18-SSX by engaging with transcriptional activators. Variant Polycomb repressive complex 1.1 (PRC1.1) acts as the main depositor of H2AK119ub1 and is therefore required for SS18-SSX occupancy. Importantly, the SSX C terminus not only depends on H2AK119ub1 for localization, but also further increases it by promoting PRC1.1 complex stability. Consequently, high H2AK119ub1 levels are a feature of murine and human synovial sarcomas. These results uncover a critical role for SSX-C in mediating gene deregulation in synovial sarcoma by providing specificity to chromatin and further enabling oncofusion binding by enhancing PRC1.1 stability and H2AK119ub1 deposition.

Situated Visual Analysis and Live Monitoring for Manufacturing.

Modern machines continuously log status reports over long periods of time, which are valuable data to optimize working routines. Data visualization is a commonly used tool to gain insights into these data, mostly in retrospective (e.g., to determine causal dependencies between the faults of different machines). We present an approach to bring such visual analyses to the shop floor to support reacting to faults in real time. This approach combines spatio-temporal analyses of time series using a handheld touch device with augmented reality for live monitoring. Important information augments machines directly in their real-world context, and detailed logs of current and historical events are displayed on the handheld device. In collaboration with an industry partner, we designed and tested our approach on a live production line to obtain feedback from operators. We compare our approach for monitoring and analysis with existing solutions that are currently deployed.

Interoperability of RTN1A in dendrite dynamics and immune functions in human Langerhans cells.

Skin is an active immune organ where professional antigen-presenting cells such as epidermal Langerhans cells (LCs) link innate and adaptive immune responses. While Reticulon 1A (RTN1A) was recently identified in LCs and dendritic cells in cutaneous and lymphoid tissues of humans and mice, its function is still unclear. Here, we studied the involvement of this protein in cytoskeletal remodeling and immune responses toward pathogens by stimulation of Toll-like receptors (TLRs) in resident LCs (rLCs) and emigrated LCs (eLCs) in human epidermis ex vivo and in a transgenic THP-1 RTN1A+ cell line. Hampering RTN1A functionality through an inhibitory antibody induced significant dendrite retraction of rLCs and inhibited their emigration. Similarly, expression of RTN1A in THP-1 cells significantly altered their morphology, enhanced aggregation potential, and inhibited the Ca2+ flux. Differentiated THP-1 RTN1A+ macrophages exhibited long cell protrusions and a larger cell body size in comparison to wild-type cells. Further, stimulation of epidermal sheets with bacterial lipoproteins (TLR1/2 and TLR2 agonists) and single-stranded RNA (TLR7 agonist) resulted in the formation of substantial clusters of rLCs and a significant decrease of RTN1A expression in eLCs. Together, our data indicate involvement of RTN1A in dendrite dynamics and structural plasticity of primary LCs. Moreover, we discovered a relation between activation of TLRs, clustering of LCs, and downregulation of RTN1A within the epidermis, thus indicating an important role of RTN1A in LC residency and maintaining tissue homeostasis.

A Role for SMARCB1 in Synovial Sarcomagenesis Reveals That SS18-SSX Induces Canonical BAF Destruction.

Reduced protein levels of SMARCB1 (also known as BAF47, INI1, SNF5) have long been observed in synovial sarcoma. Here, we show that combined Smarcb1 genetic loss with SS18-SSX expression in mice synergized to produce aggressive tumors with histomorphology, transcriptomes, and genome-wide BAF-family complex distributions distinct from SS18-SSX alone, indicating a defining role for SMARCB1 in synovial sarcoma. Smarcb1 silencing alone in mesenchyme modeled epithelioid sarcomagenesis. In mouse and human synovial sarcoma cells, SMARCB1 was identified within PBAF and canonical BAF (CBAF) complexes, coincorporated with SS18-SSX in the latter. Recombinant expression of CBAF components in human cells reconstituted CBAF subcomplexes that contained equal levels of SMARCB1 regardless of SS18 or SS18-SSX inclusion. In vivo, SS18-SSX expression led to whole-complex CBAF degradation, rendering increases in the relative prevalence of other BAF-family subtypes, PBAF and GBAF complexes, over time. Thus, SS18-SSX alters BAF subtypes levels/balance and genome distribution, driving synovial sarcomagenesis. SIGNIFICANCE: The protein level of BAF component SMARCB1 is reduced in synovial sarcoma but plays a defining role, incorporating into PBAF and SS18-SSX-containing canonical BAF complexes. Reduced levels of SMARCB1 derive from whole-complex degradation of canonical BAF driven by SS18-SSX, with relative increases in the abundance of other BAF-family subtypes.See related commentary by Maxwell and Hargreaves, p. 2375.This article is highlighted in the In This Issue feature, p. 2355.

Multi-omics reveals clinically relevant proliferative drive associated with mTOR-MYC-OXPHOS activity in chronic lymphocytic leukemia.

Chronic Lymphocytic Leukemia (CLL) has a complex pattern of driver mutations and much of its clinical diversity remains unexplained. We devised a method for simultaneous subgroup discovery across multiple data types and applied it to genomic, transcriptomic, DNA methylation and ex-vivo drug response data from 217 Chronic Lymphocytic Leukemia (CLL) cases. We uncovered a biological axis of heterogeneity strongly associated with clinical behavior and orthogonal to the known biomarkers. We validated its presence and clinical relevance in four independent cohorts (n=547 patients). We find that this axis captures the proliferative drive (PD) of CLL cells, as it associates with lymphocyte doubling rate, global hypomethylation, accumulation of driver aberrations and response to pro-proliferative stimuli. CLL-PD was linked to the activation of mTOR-MYC-oxidative phosphorylation (OXPHOS) through transcriptomic, proteomic and single cell resolution analysis. CLL-PD is a key determinant of disease outcome in CLL. Our multi-table integration approach may be applicable to other tumors whose inter-individual differences are currently unexplained.

Survey of ex vivo drug combination effects in chronic lymphocytic leukemia reveals synergistic drug effects and genetic dependencies.

Drug combinations that target critical pathways are a mainstay of cancer care. To improve current approaches to combination treatment of chronic lymphocytic leukemia (CLL) and gain insights into the underlying biology, we studied the effect of 352 drug combination pairs in multiple concentrations by analysing ex vivo drug response of 52 primary CLL samples, which were characterized by "omics" profiling. Known synergistic interactions were confirmed for B-cell receptor (BCR) inhibitors with Bcl-2 inhibitors and with chemotherapeutic drugs, suggesting that this approach can identify clinically useful combinations. Moreover, we uncovered synergistic interactions between BCR inhibitors and afatinib, which we attribute to BCR activation by afatinib through BLK upstream of BTK and PI3K. Combinations of multiple inhibitors of BCR components (e.g., BTK, PI3K, SYK) had effects similar to the single agents. While PI3K and BTK inhibitors produced overall similar effects in combinations with other drugs, we uncovered a larger response heterogeneity of combinations including PI3K inhibitors, predominantly in CLL with mutated IGHV, which we attribute to the target's position within the BCR-signaling pathway. Taken together, our study shows that drug combination effects can be effectively queried in primary cancer cells, which could aid discovery, triage and clinical development of drug combinations.

Drug-perturbation-based stratification of blood cancer.

As new generations of targeted therapies emerge and tumor genome sequencing discovers increasingly comprehensive mutation repertoires, the functional relationships of mutations to tumor phenotypes remain largely unknown. Here, we measured ex vivo sensitivity of 246 blood cancers to 63 drugs alongside genome, transcriptome, and DNA methylome analysis to understand determinants of drug response. We assembled a primary blood cancer cell encyclopedia data set that revealed disease-specific sensitivities for each cancer. Within chronic lymphocytic leukemia (CLL), responses to 62% of drugs were associated with 2 or more mutations, and linked the B cell receptor (BCR) pathway to trisomy 12, an important driver of CLL. Based on drug responses, the disease could be organized into phenotypic subgroups characterized by exploitable dependencies on BCR, mTOR, or MEK signaling and associated with mutations, gene expression, and DNA methylation. Fourteen percent of CLLs were driven by mTOR signaling in a non-BCR-dependent manner. Multivariate modeling revealed immunoglobulin heavy chain variable gene (IGHV) mutation status and trisomy 12 as the most important modulators of response to kinase inhibitors in CLL. Ex vivo drug responses were associated with outcome. This study overcomes the perception that most mutations do not influence drug response of cancer, and points to an updated approach to understanding tumor biology, with implications for biomarker discovery and cancer care.

Comprehensive long-term outcome of best drug treatment with or without add-on vagus nerve stimulation for epilepsy: a retrospective matched pairs case-control study.

PURPOSE: In the treatment of epilepsy, the recommendation to add vagus nerve stimulation (VNS) to the best available drug therapy (BDT) mostly relies on uncontrolled studies which provide limited information about VNS-specific benefits. We report findings from a retrospective matched pairs case-control study comparing the long-term (>2 years) outcomes of BDT with or without VNS. METHODS: Included were adult patients with therapy-refractory epilepsy who had undergone the pre-surgical work-up (baseline) and subsequently received BDT with VNS (BDT+VNS) or BDT alone (BDT group). Patients were matched in pairs for age, gender and follow-up. Health outcomes were assessed at least 24 months after the baseline by comprehensive postal surveys and included established psychometric scales. RESULTS: We obtained data from 20 matched pairs of case and control patients. In both groups, seizures, health-related quality of life and mood improved over time. More BDT patients experienced a complete cessation of "major" seizures (12/20 vs. 4/20) whereas, in non-seizure free patients, BDT+VNS patients showed better seizure frequency reduction (>50% reduction: 12/19 vs. 7/16). BDT+VNS patients experienced equal drug related and additional VNS related side effects. No clinically relevant effect of VNS treatment was found on any psychological/psychosocial outcome measure. CONCLUSION: This retrospective study provided no positive evidence for therapeutic benefits of adding VNS to BDT. The follow-up health status of BDT+VNS patients was slightly worse than in patients receiving BDT alone. Despite minor group differences at baseline the two patient groups who had failed presurgical evaluation were comparable. Therapeutic improvements during long-term BDT alone are often underestimated resulting in a misattribution of positive changes to VNS in uncontrolled studies and reviews. Currently, there is no incontrovertible evidence for the clinical effectiveness of adding VNS to BDT.