Proteogenetic drug response profiling elucidates targetable vulnerabilities of myelofibrosis.

Myelofibrosis is a hematopoietic stem cell disorder belonging to the myeloproliferative neoplasms. Myelofibrosis patients frequently carry driver mutations in either JAK2 or Calreticulin (CALR) and have limited therapeutic options. Here, we integrate ex vivo drug response and proteotype analyses across myelofibrosis patient cohorts to discover targetable vulnerabilities and associated therapeutic strategies. Drug sensitivities of mutated and progenitor cells were measured in patient blood using high-content imaging and single-cell deep learning-based analyses. Integration with matched molecular profiling revealed three targetable vulnerabilities. First, CALR mutations drive BET and HDAC inhibitor sensitivity, particularly in the absence of high Ras pathway protein levels. Second, an MCM complex-high proliferative signature corresponds to advanced disease and sensitivity to drugs targeting pro-survival signaling and DNA replication. Third, homozygous CALR mutations result in high endoplasmic reticulum (ER) stress, responding to ER stressors and unfolded protein response inhibition. Overall, our integrated analyses provide a molecularly motivated roadmap for individualized myelofibrosis patient treatment.

Epigenetic neural glioblastoma enhances synaptic integration and predicts therapeutic vulnerability.

Neural-tumor interactions drive glioma growth as evidenced in preclinical models, but clinical validation is nascent. We present an epigenetically defined neural signature of glioblastoma that independently affects patients' survival. We use reference signatures of neural cells to deconvolve tumor DNA and classify samples into low- or high-neural tumors. High-neural glioblastomas exhibit hypomethylated CpG sites and upregulation of genes associated with synaptic integration. Single-cell transcriptomic analysis reveals high abundance of stem cell-like malignant cells classified as oligodendrocyte precursor and neural precursor cell-like in high-neural glioblastoma. High-neural glioblastoma cells engender neuron-to-glioma synapse formation in vitro and in vivo and show an unfavorable survival after xenografting. In patients, a high-neural signature associates with decreased survival as well as increased functional connectivity and can be detected via DNA analytes and brain-derived neurotrophic factor in plasma. Our study presents an epigenetically defined malignant neural signature in high-grade gliomas that is prognostically relevant.

Targeting the Siglec-sialic acid axis promotes antitumor immune responses in preclinical models of glioblastoma.

Glioblastoma (GBM) is the most aggressive form of primary brain tumor, for which effective therapies are urgently needed. Cancer cells are capable of evading clearance by phagocytes such as microglia- and monocyte-derived cells through engaging tolerogenic programs. Here, we found that high expression of sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) correlates with reduced survival in patients with GBM. Using microglia- and monocyte-derived cell-specific knockouts of Siglec-E, the murine functional homolog of Siglec-9, together with single-cell RNA sequencing, we demonstrated that Siglec-E inhibits phagocytosis by these cells, thereby promoting immune evasion. Loss of Siglec-E on monocyte-derived cells further enhanced antigen cross-presentation and production of pro-inflammatory cytokines, which resulted in more efficient T cell priming. This bridging of innate and adaptive responses delayed tumor growth and resulted in prolonged survival in murine models of GBM. Furthermore, we showed the combinatorial activity of Siglec-E blockade and other immunotherapies demonstrating the potential for targeting Siglec-9 as a treatment for patients with GBM.

Ex vivo drug response heterogeneity reveals personalized therapeutic strategies for patients with multiple myeloma.

Multiple myeloma (MM) is a plasma cell malignancy defined by complex genetics and extensive patient heterogeneity. Despite a growing arsenal of approved therapies, MM remains incurable and in need of guidelines to identify effective personalized treatments. Here, we survey the ex vivo drug and immunotherapy sensitivities across 101 bone marrow samples from 70 patients with MM using multiplexed immunofluorescence, automated microscopy and deep-learning-based single-cell phenotyping. Combined with sample-matched genetics, proteotyping and cytokine profiling, we map the molecular regulatory network of drug sensitivity, implicating the DNA repair pathway and EYA3 expression in proteasome inhibitor sensitivity and major histocompatibility complex class II expression in the response to elotuzumab. Globally, ex vivo drug sensitivity associated with bone marrow microenvironmental signatures reflecting treatment stage, clonality and inflammation. Furthermore, ex vivo drug sensitivity significantly stratified clinical treatment responses, including to immunotherapy. Taken together, our study provides molecular and actionable insights into diverse treatment strategies for patients with MM.

ROS Induction Targets Persister Cancer Cells with Low Metabolic Activity in NRAS-Mutated Melanoma.

Clinical management of melanomas with NRAS mutations is challenging. Targeting MAPK signaling is only beneficial to a small subset of patients due to resistance that arises through genetic, transcriptional, and metabolic adaptation. Identification of targetable vulnerabilities in NRAS-mutated melanoma could help improve patient treatment. Here, we used multiomics analyses to reveal that NRAS-mutated melanoma cells adopt a mesenchymal phenotype with a quiescent metabolic program to resist cellular stress induced by MEK inhibition. The metabolic alterations elevated baseline reactive oxygen species (ROS) levels, leading these cells to become highly sensitive to ROS induction. In vivo xenograft experiments and single-cell RNA sequencing demonstrated that intratumor heterogeneity necessitates the combination of a ROS inducer and a MEK inhibitor to inhibit both tumor growth and metastasis. Ex vivo pharmacoscopy of 62 human metastatic melanomas confirmed that MEK inhibitor-resistant tumors significantly benefited from the combination therapy. Finally, oxidative stress response and translational suppression corresponded with ROS-inducer sensitivity in 486 cancer cell lines, independent of cancer type. These findings link transcriptional plasticity to a metabolic phenotype that can be inhibited by ROS inducers in melanoma and other cancers. SIGNIFICANCE: Metabolic reprogramming in drug-resistant NRAS-mutated melanoma cells confers sensitivity to ROS induction, which suppresses tumor growth and metastasis in combination with MAPK pathway inhibitors.

A functional analysis of 180 cancer cell lines reveals conserved intrinsic metabolic programs.

Cancer cells reprogram their metabolism to support growth and invasion. While previous work has highlighted how single altered reactions and pathways can drive tumorigenesis, it remains unclear how individual changes propagate at the network level and eventually determine global metabolic activity. To characterize the metabolic lifestyle of cancer cells across pathways and genotypes, we profiled the intracellular metabolome of 180 pan-cancer cell lines grown in identical conditions. For each cell line, we estimated activity for 49 pathways spanning the entirety of the metabolic network. Upon clustering, we discovered a convergence into only two major metabolic types. These were functionally confirmed by 13 C-flux analysis, lipidomics, and analysis of sensitivity to perturbations. They revealed that the major differences in cancers are associated with lipid, TCA cycle, and carbohydrate metabolism. Thorough integration of these types with multiomics highlighted little association with genetic alterations but a strong association with markers of epithelial-mesenchymal transition. Our analysis indicates that in absence of variations imposed by the microenvironment, cancer cells adopt distinct metabolic programs which serve as vulnerabilities for therapy.

Multiplexed high-throughput immune cell imaging reveals molecular health-associated phenotypes.

Phenotypic plasticity is essential to the immune system, yet the factors that shape it are not fully understood. Here, we comprehensively analyze immune cell phenotypes including morphology across human cohorts by single-round multiplexed immunofluorescence, automated microscopy, and deep learning. Using the uncertainty of convolutional neural networks to cluster the phenotypes of eight distinct immune cell subsets, we find that the resulting maps are influenced by donor age, gender, and blood pressure, revealing distinct polarization and activation-associated phenotypes across immune cell classes. We further associate T cell morphology to transcriptional state based on their joint donor variability and validate an inflammation-associated polarized T cell morphology and an age-associated loss of mitochondria in CD4+ T cells. Together, we show that immune cell phenotypes reflect both molecular and personal health information, opening new perspectives into the deep immune phenotyping of individual people in health and disease.

Deep Morphology Learning Enhances Ex Vivo Drug Profiling-Based Precision Medicine.

Drug testing in patient biopsy-derived cells can identify potent treatments for patients suffering from relapsed or refractory hematologic cancers. Here we investigate the use of weakly supervised deep learning on cell morphologies (DML) to complement diagnostic marker-based identification of malignant and nonmalignant cells in drug testing. Across 390 biopsies from 289 patients with diverse blood cancers, DML-based drug responses show improved reproducibility and clustering of drugs with the same mode of action. DML does so by adapting to batch effects and by autonomously recognizing disease-associated cell morphologies. In a post hoc analysis of 66 patients, DML-recommended treatments led to improved progression-free survival compared with marker-based recommendations and physician's choice-based treatments. Treatments recommended by both immunofluorescence and DML doubled the fraction of patients achieving exceptional clinical responses. Thus, DML-enhanced ex vivo drug screening is a promising tool in the identification of effective personalized treatments. SIGNIFICANCE: We have recently demonstrated that image-based drug screening in patient samples identifies effective treatment options for patients with advanced blood cancers. Here we show that using deep learning to identify malignant and nonmalignant cells by morphology improves such screens. The presented workflow is robust, automatable, and compatible with clinical routine. This article is highlighted in the In This Issue feature, p. 476.

Multifunctional mRNA-Based CAR T Cells Display Promising Antitumor Activity Against Glioblastoma.

PURPOSE: Most chimeric antigen receptor (CAR) T-cell strategies against glioblastoma have demonstrated only modest therapeutic activity and are based on persistent gene modification strategies that have limited transgene capacity, long manufacturing processes, and the risk for uncontrollable off-tumor toxicities. mRNA-based T-cell modifications are an emerging safe, rapid, and cost-effective alternative to overcome these challenges, but are underexplored against glioblastoma. EXPERIMENTAL DESIGN: We generated mouse and human mRNA-based multifunctional T cells coexpressing a multitargeting CAR based on the natural killer group 2D (NKG2D) receptor and the proinflammatory cytokines IL12 and IFNα2 and assessed their antiglioma activity in vitro and in vivo. RESULTS: Compared with T cells that either expressed the CAR or cytokines alone, multifunctional CAR T cells demonstrated increased antiglioma activity in vitro and in vivo in three orthotopic immunocompetent mouse glioma models without signs of toxicity. Mechanistically, the coexpression of IL12 and IFNα2 in addition to the CAR promoted a proinflammatory tumor microenvironment and reduced T-cell exhaustion as demonstrated by ex vivo immune phenotyping, cytokine profiling, and RNA sequencing. The translational potential was demonstrated by image-based single-cell analyses of mRNA-modified T cells in patient glioblastoma samples with a complex cellular microenvironment. This revealed strong antiglioma activity of human mRNA-based multifunctional NKG2D CAR T cells coexpressing IL12 and IFNα2 whereas T cells that expressed either the CAR or cytokines alone did not demonstrate comparable antiglioma activity. CONCLUSIONS: These data provide a robust rationale for future clinical studies with mRNA-based multifunctional CAR T cells to treat malignant brain tumors.

A physical wiring diagram for the human immune system.

The human immune system is composed of a distributed network of cells circulating throughout the body, which must dynamically form physical associations and communicate using interactions between their cell-surface proteomes1. Despite their therapeutic potential2, our map of these surface interactions remains incomplete3,4. Here, using a high-throughput surface receptor screening method, we systematically mapped the direct protein interactions across a recombinant library that encompasses most of the surface proteins that are detectable on human leukocytes. We independently validated and determined the biophysical parameters of each novel interaction, resulting in a high-confidence and quantitative view of the receptor wiring that connects human immune cells. By integrating our interactome with expression data, we identified trends in the dynamics of immune interactions and constructed a reductionist mathematical model that predicts cellular connectivity from basic principles. We also developed an interactive multi-tissue single-cell atlas that infers immune interactions throughout the body, revealing potential functional contexts for new interactions and hubs in multicellular networks. Finally, we combined targeted protein stimulation of human leukocytes with multiplex high-content microscopy to link our receptor interactions to functional roles, in terms of both modulating immune responses and maintaining normal patterns of intercellular associations. Together, our work provides a systematic perspective on the intercellular wiring of the human immune system that extends from systems-level principles of immune cell connectivity down to mechanistic characterization of individual receptors, which could offer opportunities for therapeutic intervention.

Light-mediated discovery of surfaceome nanoscale organization and intercellular receptor interaction networks.

The molecular nanoscale organization of the surfaceome is a fundamental regulator of cellular signaling in health and disease. Technologies for mapping the spatial relationships of cell surface receptors and their extracellular signaling synapses would unlock theranostic opportunities to target protein communities and the possibility to engineer extracellular signaling. Here, we develop an optoproteomic technology termed LUX-MS that enables the targeted elucidation of acute protein interactions on and in between living cells using light-controlled singlet oxygen generators (SOG). By using SOG-coupled antibodies, small molecule drugs, biologics and intact viral particles, we demonstrate the ability of LUX-MS to decode ligand receptor interactions across organisms and to discover surfaceome receptor nanoscale organization with direct implications for drug action. Furthermore, by coupling SOG to antigens we achieved light-controlled molecular mapping of intercellular signaling within functional immune synapses between antigen-presenting cells and CD8+ T cells providing insights into T cell activation with spatiotemporal specificity. LUX-MS based decoding of surfaceome signaling architectures thereby provides a molecular framework for the rational development of theranostic strategies.

Allosteric Antagonist Modulation of TRPV2 by Piperlongumine Impairs Glioblastoma Progression.

The use of computational tools to identify biological targets of natural products with anticancer properties and unknown modes of action is gaining momentum. We employed self-organizing maps to deconvolute the phenotypic effects of piperlongumine (PL) and establish a link to modulation of the human transient receptor potential vanilloid 2 (hTRPV2) channel. The structure of the PL-bound full-length rat TRPV2 channel was determined by cryo-EM. PL binds to a transient allosteric pocket responsible for a new mode of anticancer activity against glioblastoma (GBM) in which hTRPV2 is overexpressed. Calcium imaging experiments revealed the importance of Arg539 and Thr522 residues on the antagonistic effect of PL and calcium influx modulation of the TRPV2 channel. Downregulation of hTRPV2 reduces sensitivity to PL and decreases ROS production. Analysis of GBM patient samples associates hTRPV2 overexpression with tumor grade, disease progression, and poor prognosis. Extensive tumor abrogation and long term survival was achieved in two murine models of orthotopic GBM by formulating PL in an implantable scaffold/hydrogel for sustained local therapy. Furthermore, in primary tumor samples derived from GBM patients, we observed a selective reduction of malignant cells in response to PL ex vivo. Our results establish a broadly applicable strategy, leveraging data-motivated research hypotheses for the discovery of novel means tackling cancer.

Beneficial Metabolic Effects of TREM2 in Obesity Are Uncoupled From Its Expression on Macrophages.

Obesity-induced white adipose tissue (WAT) hypertrophy is associated with elevated adipose tissue macrophage (ATM) content. Overexpression of the triggering receptor expressed on myeloid cells 2 (TREM2) reportedly increases adiposity, worsening health. Paradoxically, using insulin resistance, elevated fat mass, and hypercholesterolemia as hallmarks of unhealthy obesity, a recent report demonstrated that ATM-expressed TREM2 promoted health. Here, we identified that in mice, TREM2 deficiency aggravated diet-induced insulin resistance and hepatic steatosis independently of fat and cholesterol levels. Metabolomics linked TREM2 deficiency with elevated obesity-instigated serum ceramides that correlated with impaired insulin sensitivity. Remarkably, while inhibiting ceramide synthesis exerted no influences on TREM2-dependent ATM remodeling, inflammation, or lipid load, it restored insulin tolerance, reversing adipose hypertrophy and secondary hepatic steatosis of TREM2-deficient animals. Bone marrow transplantation experiments revealed unremarkable influences of immune cell-expressed TREM2 on health, instead demonstrating that WAT-intrinsic mechanisms impinging on sphingolipid metabolism dominate in the systemic protective effects of TREM2 on metabolic health.

The Tumor Profiler Study: integrated, multi-omic, functional tumor profiling for clinical decision support.

The application and integration of molecular profiling technologies create novel opportunities for personalized medicine. Here, we introduce the Tumor Profiler Study, an observational trial combining a prospective diagnostic approach to assess the relevance of in-depth tumor profiling to support clinical decision-making with an exploratory approach to improve the biological understanding of the disease.

Kinase Interaction Network Expands Functional and Disease Roles of Human Kinases.

Protein kinases are essential for signal transduction and control of most cellular processes, including metabolism, membrane transport, motility, and cell cycle. Despite the critical role of kinases in cells and their strong association with diseases, good coverage of their interactions is available for only a fraction of the 535 human kinases. Here, we present a comprehensive mass-spectrometry-based analysis of a human kinase interaction network covering more than 300 kinases. The interaction dataset is a high-quality resource with more than 5,000 previously unreported interactions. We extensively characterized the obtained network and were able to identify previously described, as well as predict new, kinase functional associations, including those of the less well-studied kinases PIM3 and protein O-mannose kinase (POMK). Importantly, the presented interaction map is a valuable resource for assisting biomedical studies. We uncover dozens of kinase-disease associations spanning from genetic disorders to complex diseases, including cancer.

Common Nodes of Virus-Host Interaction Revealed Through an Integrated Network Analysis.

Viruses are one of the major causes of acute and chronic infectious diseases and thus a major contributor to the global burden of disease. Several studies have shown how viruses have evolved to hijack basic cellular pathways and evade innate immune response by modulating key host factors and signaling pathways. A collective view of these multiple studies could advance our understanding of virus-host interactions and provide new therapeutic perspectives for the treatment of viral diseases. Here, we performed an integrative meta-analysis to elucidate the 17 different host-virus interactomes. Network and bioinformatics analyses showed how viruses with small genomes efficiently achieve the maximal effect by targeting multifunctional and highly connected host proteins with a high occurrence of disordered regions. We also identified the core cellular process subnetworks that are targeted by all the viruses. Integration with functional RNA interference (RNAi) datasets showed that a large proportion of the targets are required for viral replication. Furthermore, we performed an interactome-informed drug re-purposing screen and identified novel activities for broad-spectrum antiviral agents against hepatitis C virus and human metapneumovirus. Altogether, these orthogonal datasets could serve as a platform for hypothesis generation and follow-up studies to broaden our understanding of the viral evasion landscape.

Combined chemosensitivity and chromatin profiling prioritizes drug combinations in CLL.

The Bruton tyrosine kinase (BTK) inhibitor ibrutinib has substantially improved therapeutic options for chronic lymphocytic leukemia (CLL). Although ibrutinib is not curative, it has a profound effect on CLL cells and may create new pharmacologically exploitable vulnerabilities. To identify such vulnerabilities, we developed a systematic approach that combines epigenome profiling (charting the gene-regulatory basis of cell state) with single-cell chemosensitivity profiling (quantifying cell-type-specific drug response) and bioinformatic data integration. By applying our method to a cohort of matched patient samples collected before and during ibrutinib therapy, we identified characteristic ibrutinib-induced changes that provide a starting point for the rational design of ibrutinib combination therapies. Specifically, we observed and validated preferential sensitivity to proteasome, PLK1, and mTOR inhibitors during ibrutinib treatment. More generally, our study establishes a broadly applicable method for investigating treatment-specific vulnerabilities by integrating the complementary perspectives of epigenetic cell states and phenotypic drug responses in primary patient samples.

Human "TH9" cells are a subpopulation of PPAR-γ+ TH2 cells.

Although TH1, TH2, and TH17 cells are well-defined TH cell lineages in humans, it remains debated whether IL-9-producing TH cells represent a bona fide "TH9" lineage. Our understanding of the cellular characteristics and functions of IL-9-producing TH cells in humans is still nascent. Here, we report that human IL-9-producing TH cells express the chemokine receptors CCR4 and CCR8, produce high levels of IL-5 and IL-13, and express TH2 lineage-associated transcription factors. In these cells, IL-9 production is activation dependent, transient, and accompanied by down-regulation of TH2 cytokines, leading to an apparent "TH9" phenotype. IL-9+ TH2 cells can be distinguished from "conventional" TH2 cells based on their expression of the transcription factor PPAR-γ. Accordingly, PPAR-γ is induced in naïve TH cells by priming with IL-4 and TGF-ß ("TH9" priming) and is required for IL-9 production. In line with their identity as early activated TH2 cells, IL-9+ TH2 cells are found in acute allergic skin inflammation in humans. We propose that IL-9-producing TH cells are a phenotypically and functionally distinct subpopulation of TH2 cells that depend on PPAR-γ for full effector functions.

A novel dual-cytokine-antibody fusion protein for the treatment of CD38-positive malignancies.

A novel dual-cytokine-antibody fusion protein, consisting of an antibody directed against CD38 [a tumor-associated antigen mainly expressed on the surface of multiple myeloma (MM) cells], simultaneously fused to both tumor necrosis factor ligand superfamily member 10 (TRAIL) and interleukin-2 (IL2), was designed, expressed and purified to homogeneity. The novel fusion protein, termed IL2-αCD38-αCD38-scTRAIL, was able to selectively recognize its cognate antigen expressed on the surface of MM and lymphoma cell lines, as evidenced by flow cytometry analysis. Moreover, the targeted version of TRAIL was able to induce cancer cell death in vitro, both with MM cell lines and with fresh isolates from the bone marrow of MM patients. The experiments provide a rationale for possible future applications of IL2-αCD38-αCD38-scTRAIL for the treatment of patients with MM or other CD38-positive malignancies.