T cells with increased responsiveness cause obesity in mice without diet intervention.

Obesity is a complex multicausal disease that can cause morbidity and mortality, and there is need for improved knowledge on the underlying mechanisms. Using a mouse model of increased T cell responsiveness, we show that development of obesity can be driven by immune cells. This was confirmed with bone marrow transplantation and adoptive T cell transfer to several recipient mouse models. Single-cell RNA sequencing and CyTOF analysis showed that the mice display altered composition of circulating T cells and increased T cell activation in visceral adipose tissue, suggesting activated T cells as critical players in the increased fat mass. In this study, we provide evidence that obesity can be driven by immune cell activity and in particular by T cells, which could have broad implications for prevention and treatment of this condition.

Evolutionary mode and timing of dissemination of high-grade serous carcinomas.

Dissemination within the peritoneal cavity is a main determinant of poor patient outcomes from high-grade serous carcinomas (HGSCs). The dissemination process is poorly understood from a cancer evolutionary perspective. We reconstructed the evolutionary trajectories across a median of 5 tumor sites and regions from each of 23 patients based on deep whole-exome sequencing. Polyclonal cancer origin was detected in 1 patient. Ovarian tumors had more complex subclonal architectures than other intraperitoneal tumors in each patient, which indicated that tumors developed earlier in the ovaries. Three common modes of dissemination were identified, including monoclonal or polyclonal dissemination of monophyletic (linear) or polyphyletic (branched) subclones. Mutation profiles of initial or disseminated clones varied greatly among cancers, but recurrent mutations were found in 7 cancer-critical genes, including TP53, BRCA1, BRCA2, and DNMT3A, and in the PI3K/AKT1 pathway. Disseminated clones developed late in the evolutionary trajectory models of most cancers, in particular in cancers with DNA damage repair deficiency. Polyclonal dissemination was predicted to occur predominantly as a single and rapid wave, but chemotherapy exposure was associated with higher genomic diversity of disseminated clones. In conclusion, we described three common evolutionary dissemination modes across HGSCs and proposed factors associated with dissemination diversity.

Small molecule inhibitors targeting regulatory T cells for cancer treatment.

Regulatory T cells (Tregs) are important controllers of the immune system homeostasis by preventing disproportionate immune responses. In the context of cancer, Tregs contribute to tumor development by suppressing other immune cells in the tumor microenvironment (TME). Infiltration of Tregs in the TME has been associated with poor prognosis in cancer patients. Thus, understanding the mechanisms underlying Treg recruitment and suppressive functions is essential for developing cancer immunotherapies to boost antitumor immune responses. While antibody-based strategies targeting Tregs have shown promise, small molecule inhibitors offer distinct advantages, including oral bioavailability and the ability to penetrate the TME and target intracellular proteins. Here, we provide an overview of small molecule inhibitors that have demonstrated efficacy in modulating Tregs activity in cancer and highlight the need for phenotypic assays to characterize therapeutic compounds.

Standardized assays to monitor drug sensitivity in hematologic cancers.

The principle of drug sensitivity testing is to expose cancer cells to a library of different drugs and measure its effects on cell viability. Recent technological advances, continuous approval of targeted therapies, and improved cell culture protocols have enhanced the precision and clinical relevance of such screens. Indeed, drug sensitivity testing has proven diagnostically valuable for patients with advanced hematologic cancers. However, different cell types behave differently in culture and therefore require optimized drug screening protocols to ensure that their ex vivo drug sensitivity accurately reflects in vivo drug responses. For example, primary chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) cells require unique microenvironmental stimuli to survive in culture, while this is less the case for acute myeloid leukemia (AML) cells. Here, we present our optimized and validated protocols for culturing and drug screening of primary cells from AML, CLL, and MM patients, and a generic protocol for cell line models. We also discuss drug library designs, reproducibility, and quality controls. We envision that these protocols may serve as community guidelines for the use and interpretation of assays to monitor drug sensitivity in hematologic cancers and thus contribute to standardization. The read-outs may provide insight into tumor biology, identify or confirm treatment resistance and sensitivity in real time, and ultimately guide clinical decision-making.

Modified study designs to expand treatment options in personalised oncology: a multistakeholder view.

Personalised oncology, whereby patients are given therapies based on their molecular tumour profile, is rapidly becoming an essential part of optimal clinical care, at least partly facilitated by recent advances in next-generation sequencing-based technology using liquid- and tissue-based biopsies. Consequently, clinical trials have shifted in approach, from traditional studies evaluating cytotoxic chemotherapy in largely histology-based populations to modified, biomarker-driven studies (e.g. basket, umbrella, platform) of molecularly guided therapies and cancer immunotherapies in selected patient subsets. Such modified study designs may assess, within the same trial structure, multiple cancer types and treatments, and should incorporate a multistakeholder perspective. This is key to generating complementary, fit-for-purpose and timely evidence for molecularly guided therapies that can be used as proof-of-concept to inform further study designs, lead to approval by regulatory authorities and be used as confirmation of clinical benefit for health technology assessment bodies. In general, the future of cancer clinical trials requires a framework for the application of innovative technologies and dynamic design methodologies, in order to efficiently transform scientific discoveries into clinical utility. Next-generation, modified studies that involve the joint efforts of all key stakeholders will offer individualised strategies that ultimately contribute to globalised knowledge and collective learning. In this review, we outline the background and purpose of such modified study designs and detail key aspects from a multistakeholder perspective. We also provide methodological considerations for designing the studies and highlight how insights from already-ongoing studies may address current challenges and opportunities in the era of personalised oncology.

Diversity of intratumoral regulatory T cells in B-cell non-Hodgkin lymphoma.

Tumor-infiltrating regulatory T cells (Tregs) contribute to an immunosuppressive tumor microenvironment. Despite extensive studies, the prognostic impact of tumor-infiltrating Tregs in B-cell non-Hodgkin lymphomas (B-NHLs) remains unclear. Emerging studies suggest substantial heterogeneity in the phenotypes and suppressive capacities of Tregs, emphasizing the importance of understanding Treg diversity and the need for additional markers to identify highly suppressive Tregs. Here, we applied single-cell RNA sequencing and T-cell receptor sequencing combined with high-dimensional cytometry to decipher the heterogeneity of intratumoral Tregs in diffuse large B-cell lymphoma and follicular lymphoma (FL), compared with that in nonmalignant tonsillar tissue. We identified 3 distinct transcriptional states of Tregs: resting, activated, and unconventional LAG3+FOXP3- Tregs. Activated Tregs were enriched in B-NHL tumors, coexpressed several checkpoint receptors, and had stronger immunosuppressive activity compared with resting Tregs. In FL, activated Tregs were found in closer proximity to CD4+ and CD8+ T cells than other cell types. Furthermore, we used a computational approach to develop unique gene signature matrices, which were used to enumerate each Treg subset in cohorts with bulk gene expression data. In 2 independent FL cohorts, activated Tregs was the major subset, and high abundance was associated with adverse outcome. This study demonstrates that Tregs infiltrating B-NHL tumors are transcriptionally and functionally diverse. Highly immunosuppressive activated Tregs were enriched in tumor tissue but absent in the peripheral blood. Our data suggest that a deeper understanding of Treg heterogeneity in B-NHL could open new paths for rational drug design, facilitating selective targeting to improve antitumor immunity.

A tumor microenvironment model of chronic lymphocytic leukemia enables drug sensitivity testing to guide precision medicine.

The microenvironment of chronic lymphocytic leukemia (CLL) cells in lymph nodes, spleen, and bone marrow provides survival, proliferation, and drug resistance signals. Therapies need to be effective in these compartments, and pre-clinical models of CLL that are used to test drug sensitivity must mimic the tumor microenvironment to reflect clinical responses. Ex vivo models have been developed that capture individual or multiple aspects of the CLL microenvironment, but they are not necessarily compatible with high-throughput drug screens. Here, we report on a model that has reasonable associated costs, can be handled in a regularly equipped cell lab, and is compatible with ex vivo functional assays including drug sensitivity screens. The CLL cells are cultured with fibroblasts that express the ligands APRIL, BAFF and CD40L for 24 h. The transient co-culture was shown to support survival of primary CLL cells for at least 13 days, and mimic in vivo drug resistance signals. Ex vivo sensitivity and resistance to the Bcl-2 antagonist venetoclax correlated with in vivo responses. The assay was used to identify treatment vulnerabilities and guide precision medicine for a patient with relapsed CLL. Taken together, the presented CLL microenvironment model enables clinical implementation of functional precision medicine in CLL.

Phenotypic deconvolution in heterogeneous cancer cell populations using drug-screening data.

Tumor heterogeneity is an important driver of treatment failure in cancer since therapies often select for drug-tolerant or drug-resistant cellular subpopulations that drive tumor growth and recurrence. Profiling the drug-response heterogeneity of tumor samples using traditional genomic deconvolution methods has yielded limited results, due in part to the imperfect mapping between genomic variation and functional characteristics. Here, we leverage mechanistic population modeling to develop a statistical framework for profiling phenotypic heterogeneity from standard drug-screen data on bulk tumor samples. This method, called PhenoPop, reliably identifies tumor subpopulations exhibiting differential drug responses and estimates their drug sensitivities and frequencies within the bulk population. We apply PhenoPop to synthetically generated cell populations, mixed cell-line experiments, and multiple myeloma patient samples and demonstrate how it can provide individualized predictions of tumor growth under candidate therapies. This methodology can also be applied to deconvolution problems in a variety of biological settings beyond cancer drug response.

Disruption of Phosphodiesterase 3A Binding to SERCA2 Increases SERCA2 Activity and Reduces Mortality in Mice With Chronic Heart Failure.

BACKGROUND: Increasing SERCA2 (sarco[endo]-plasmic reticulum Ca2+ ATPase 2) activity is suggested to be beneficial in chronic heart failure, but no selective SERCA2-activating drugs are available. PDE3A (phosphodiesterase 3A) is proposed to be present in the SERCA2 interactome and limit SERCA2 activity. Disruption of PDE3A from SERCA2 might thus be a strategy to develop SERCA2 activators. METHODS: Confocal microscopy, 2-color direct stochastic optical reconstruction microscopy, proximity ligation assays, immunoprecipitations, peptide arrays, and surface plasmon resonance were used to investigate colocalization between SERCA2 and PDE3A in cardiomyocytes, map the SERCA2/PDE3A interaction sites, and optimize disruptor peptides that release PDE3A from SERCA2. Functional experiments assessing the effect of PDE3A-binding to SERCA2 were performed in cardiomyocytes and HEK293 vesicles. The effect of SERCA2/PDE3A disruption by the disruptor peptide OptF (optimized peptide F) on cardiac mortality and function was evaluated during 20 weeks in 2 consecutive randomized, blinded, and controlled preclinical trials in a total of 148 mice injected with recombinant adeno-associated virus 9 (rAAV9)-OptF, rAAV9-control (Ctrl), or PBS, before undergoing aortic banding (AB) or sham surgery and subsequent phenotyping with serial echocardiography, cardiac magnetic resonance imaging, histology, and functional and molecular assays. RESULTS: PDE3A colocalized with SERCA2 in human nonfailing, human failing, and rodent myocardium. Amino acids 277-402 of PDE3A bound directly to amino acids 169-216 within the actuator domain of SERCA2. Disruption of PDE3A from SERCA2 increased SERCA2 activity in normal and failing cardiomyocytes. SERCA2/PDE3A disruptor peptides increased SERCA2 activity also in the presence of protein kinase A inhibitors and in phospholamban-deficient mice, and had no effect in mice with cardiomyocyte-specific inactivation of SERCA2. Cotransfection of PDE3A reduced SERCA2 activity in HEK293 vesicles. Treatment with rAAV9-OptF reduced cardiac mortality compared with rAAV9-Ctrl (hazard ratio, 0.26 [95% CI, 0.11 to 0.63]) and PBS (hazard ratio, 0.28 [95% CI, 0.09 to 0.90]) 20 weeks after AB. Mice injected with rAAV9-OptF had improved contractility and no difference in cardiac remodeling compared with rAAV9-Ctrl after aortic banding. CONCLUSIONS: Our results suggest that PDE3A regulates SERCA2 activity through direct binding, independently of the catalytic activity of PDE3A. Targeting the SERCA2/PDE3A interaction prevented cardiac mortality after AB, most likely by improving cardiac contractility.

Mutational analysis and protein profiling predict drug sensitivity in multiple myeloma cell lines.

Introduction: Multiple myeloma (MM) is a heterogeneous disease where cancer-driver mutations and aberrant signaling may lead to disease progression and drug resistance. Drug responses vary greatly, and there is an unmet need for biomarkers that can guide precision cancer medicine in this disease. Methods: To identify potential predictors of drug sensitivity, we applied integrated data from drug sensitivity screening, mutational analysis and functional signaling pathway profiling in 9 cell line models of MM. We studied the sensitivity to 33 targeted drugs and their association with the mutational status of cancer-driver genes and activity level of signaling proteins. Results: We found that sensitivity to mitogen-activated protein kinase kinase 1 (MEK1) and phosphatidylinositol-3 kinase (PI3K) inhibitors correlated with mutations in NRAS/KRAS, and PI3K family genes, respectively. Phosphorylation status of MEK1 and protein kinase B (AKT) correlated with sensitivity to MEK and PI3K inhibition, respectively. In addition, we found that enhanced phosphorylation of proteins, including Tank-binding kinase 1 (TBK1), as well as high expression of B cell lymphoma 2 (Bcl-2), correlated with low sensitivity to MEK inhibitors. Discussion: Taken together, this study shows that mutational status and signaling protein profiling might be used in further studies to predict drug sensitivities and identify resistance markers in MM.

Immunoregulatory signal networks and tumor immune evasion mechanisms: insights into therapeutic targets and agents in clinical development.

Through activation of immune cells, the immune system is responsible for identifying and destroying infected or otherwise damaged cells including tumorigenic cells that can be recognized as foreign, thus maintaining homeostasis. However, tumor cells have evolved several mechanisms to avoid immune cell detection and killing, resulting in tumor growth and progression. In the tumor microenvironment, tumor infiltrating immune cells are inactivated by soluble factors or tumor promoting conditions and lose their effects on tumor cells. Analysis of signaling and crosstalk between immune cells and tumor cells have helped us to understand in more detail the mechanisms of tumor immune evasion and this forms basis for drug development strategies in the area of cancer immunotherapy. In this review, we will summarize the dominant signaling networks involved in immune escape and describe the status of development of therapeutic strategies to target tumor immune evasion mechanisms with focus on how the tumor microenvironment interacts with T cells.

Functional precision cancer medicine: drug sensitivity screening enabled by cell culture models.

Functional precision medicine is a new, emerging area that can guide cancer treatment by capturing information from direct perturbations of tumor-derived, living cells, such as by drug sensitivity screening. Precision cancer medicine as currently implemented in clinical practice has been driven by genomics, and current molecular tumor boards rely extensively on genomic characterization to advise on therapeutic interventions. However, genomic biomarkers can only guide treatment decisions for a fraction of the patients. In this review we provide an overview of the current state of functional precision medicine, highlight advances for drug-sensitivity screening enabled by cell culture models, and discuss how artificial intelligence (AI) can be coupled to functional precision medicine to guide patient stratification.

Functional Testing to Characterize and Stratify PI3K Inhibitor Responses in Chronic Lymphocytic Leukemia.

PURPOSE: PI3K inhibitors (PI3Ki) are approved for relapsed chronic lymphocytic leukemia (CLL). Although patients may show an initial response to these therapies, development of treatment intolerance or resistance remain clinical challenges. To overcome these, prediction of individual treatment responses based on actionable biomarkers is needed. Here, we characterized the activity and cellular effects of 10 PI3Ki and investigated whether functional analyses can identify treatment vulnerabilities in PI3Ki-refractory/intolerant CLL and stratify responders to PI3Ki. EXPERIMENTAL DESIGN: Peripheral blood mononuclear cell samples (n = 51 in total) from treatment-naïve and PI3Ki-treated patients with CLL were studied. Cells were profiled against 10 PI3Ki and the Bcl-2 antagonist venetoclax. Cell signaling and immune phenotypes were analyzed by flow cytometry. Cell viability was monitored by detection of cleaved caspase-3 and the CellTiter-Glo assay. RESULTS: pan-PI3Kis were most effective at inhibiting PI3K signaling and cell viability, and showed activity in CLL cells from both treatment-naïve and idelalisib-refractory/intolerant patients. CLL cells from idelalisib-refractory/intolerant patients showed overall reduced protein phosphorylation levels. The pan-PI3Ki copanlisib, but not the p110δ inhibitor idelalisib, inhibited PI3K signaling in CD4+ and CD8+ T cells in addition to CD19+ B cells, but did not significantly affect T-cell numbers. Combination treatment with a PI3Ki and venetoclax resulted in synergistic induction of apoptosis. Analysis of drug sensitivities to 73 drug combinations and profiling of 31 proteins stratified responders to idelalisib and umbralisib, respectively. CONCLUSIONS: Our findings suggest novel treatment vulnerabilities in idelalisib-refractory/intolerant CLL, and indicate that ex vivo functional profiling may stratify PI3Ki responders.

Ex vivo drug sensitivity screening in multiple myeloma identifies drug combinations that act synergistically.

The management of multiple myeloma (MM) is challenging: An assortment of available drug combinations adds complexity to treatment selection, and treatment resistance frequently develops. Given the heterogeneous nature of MM, personalized testing tools are required to identify drug sensitivities. To identify drug sensitivities in MM cells, we established a drug testing pipeline to examine ex vivo drug responses. MM cells from 44 patients were screened against 30 clinically relevant single agents and 44 double- and triple-drug combinations. We observed variability in responses across samples. The presence of gain(1q21) was associated with low sensitivity to venetoclax, and decreased ex vivo responses to dexamethasone reflected the drug resistance observed in patients. Less heterogeneity and higher efficacy was detected with many combinations compared to the corresponding single agents. We identified new synergistic effects of melflufen plus panobinostat using low concentrations (0.1-10 nm and 8 nm, respectively). In agreement with clinical studies, clinically approved combinations, such as triple combination of selinexor plus bortezomib plus dexamethasone, acted synergistically, and synergies required low drug concentrations (0.1 nm bortezomib, 10 nm selinexor and 4 nm dexamethasone). In summary, our drug screening provided results within a clinically actionable 5-day time frame and identified synergistic drug efficacies in patient-derived MM cells that may aid future therapy choices.

Mcl-1 and Bcl-xL levels predict responsiveness to dual MEK/Bcl-2 inhibition in B-cell malignancies.

Most patients with chronic lymphocytic leukemia (CLL) initially respond to targeted therapies, but eventually relapse and develop resistance. Novel treatment strategies are therefore needed to improve patient outcomes. Here, we performed direct drug testing on primary CLL cells and identified synergy between eight different mitogen-activated protein kinase kinase (MEK) inhibitors and the B-cell lymphoma 2 (Bcl-2) antagonist venetoclax. Drug sensitivity was independent of immunoglobulin heavy-chain gene variable region (IGVH) and tumor protein p53 (TP53) mutational status, and CLL cells from idelalisib-resistant patients remained sensitive to the treatment. This suggests that combined MEK/Bcl-2 inhibition may be an option for high-risk CLL. To test whether sensitivity could be detected in other B-cell malignancies, we performed drug testing on cell line models of CLL (n = 4), multiple myeloma (MM; n = 8), and mantle cell lymphoma (MCL; n = 7). Like CLL, MM cells were sensitive to the MEK inhibitor trametinib, and synergy was observed with venetoclax. In contrast, MCL cells were unresponsive to MEK inhibition. To investigate the underlying mechanisms of the disease-specific drug sensitivities, we performed flow cytometry-based high-throughput profiling of 31 signaling proteins and regulators of apoptosis in the 19 cell lines. We found that high expression of the antiapoptotic proteins myeloid cell leukemia-1 (Mcl-1) or B-cell lymphoma-extra large (Bcl-xL) predicted low sensitivity to trametinib + venetoclax. The low sensitivity could be overcome by combined treatment with an Mcl-1 or Bcl-xL inhibitor. Our findings suggest that MEK/Bcl-2 inhibition has therapeutic potential in leukemia and myeloma, and demonstrate that protein expression levels can serve as predictive biomarkers for treatment sensitivities.

A Phase I/II randomized trial of H56:IC31 vaccination and adjunctive cyclooxygenase-2-inhibitor treatment in tuberculosis patients.

Host-directed-therapy strategies are warranted to fight tuberculosis. Here we assess the safety and immunogenicity of adjunctive vaccination with the H56:IC31 candidate and cyclooxygenase-2-inhibitor treatment (etoricoxib) in pulmonary and extra-pulmonary tuberculosis patients in a randomized open-label phase I/II clinical trial (TBCOX2, NCT02503839). A total of 222 patients were screened, 51 enrolled and randomized; 13 in the etoricoxib-group, 14 in the H56:IC31-group, 12 in the etoricoxib+H56:IC31-group and 12 controls. Three Serious Adverse Events were reported in the etoricoxib-groups; two urticarial rash and one possible disease progression, no Serious Adverse Events were vaccine related. H56:IC31 induces robust expansion of antigen-specific T-cells analyzed by fluorospot and flow cytometry, and higher proportion of seroconversions. Etoricoxib reduced H56:IC31-induced T-cell responses. Here, we show the first clinical data that H56:IC31 vaccination is safe and immunogenic in tuberculosis patients, supporting further studies of H56:IC31 as a host-directed-therapy strategy. Although etoricoxib appears safe, our data do not support therapy with adjunctive cyclooxygenase-2-inhibitors.

Systems approach reveals distinct and shared signaling networks of the four PGE2 receptors in T cells.

Prostaglandin E2 (PGE2) promotes an immunosuppressive microenvironment in cancer, partly by signaling through four receptors (EP1, EP2, EP3, and EP4) on T cells. Here, we comprehensively characterized PGE2 signaling networks in helper, cytotoxic, and regulatory T cells using a phosphoproteomics and phosphoflow cytometry approach. We identified ~1500 PGE2-regulated phosphosites and several important EP1­4 signaling nodes, including PKC, CK2, PKA, PI3K, and Src. T cell subtypes exhibited distinct signaling pathways, with the strongest signaling in EP2-stimulated CD8+ cells. EP2 and EP4, both of which signal through Gαs, induced similar signaling outputs, but with distinct kinetics and intensity. Functional predictions from the observed phosphosite changes revealed PGE2 regulation of key cellular and immunological processes. Last, network modeling suggested signal integration between the receptors and a substantial contribution from G protein­independent signaling. This study offers a comprehensive view of the different PGE2-regulated phosphoproteomes in T cell subsets, providing a valuable resource for further research on this physiologically and pathophysiologically important signaling system.