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The PI3K signaling pathway regulates key cellular processes and is one of the most aberrantly activated pathways in cancer. The class I PI3K catalytic subunits p110γ and p110δ are highly enriched in leukocytes, providing an additional rationale for targeting these PI3Ks in hematologic malignancies. In 2014, the PI3Kδ inhibitor idelalisib was the first of four PI3K inhibitors (PI3Ki) to receive regulatory approval for relapsed B-cell malignancies. This was followed by approvals of the pan-class I inhibitor copanlisib (2017), the dual PI3Kγ/δ inhibitor duvelisib (2018), and the PI3Kδ and casein kinase 1ε inhibitor umbralisib (2021). Copanlisib and umbralisib received accelerated approvals, whereas idelalisib and duvelisib received initial accelerated approvals followed by full approvals. The accelerated approvals were based on overall response rates; however, follow-up studies showed increased risk of death and serious side effects. Furthermore, the confirmatory trial with copanlisib failed to show an improvement in progression-free survival when compared with chemoimmunotherapy. These developments led to black box warnings for idelalisib and duvelisib and withdrawal of copanlisib and umbralisib from the market by their manufacturers. Given the uncertain future of this drug class, additional manufacturers terminated ongoing phase III trials with novel PI3Kis. In this study, we review the development and current status of PI3Kis in hematology, limitations to their use, and our perspective on whether there is a future for PI3Kis in hematology., (©2024 American Association for Cancer Research.)

The microenvironment of hematologic cancers contributes to tumor cell survival and proliferation, as well as treatment resistance. Understanding tumor- and drug-induced changes to the immune cell composition and functionality is therefore critical for implementing optimal treatment strategies and for the development of novel cancer therapies. The liquid nature of peripheral blood makes this organ uniquely suited for single-cell studies by flow cytometry. (Phospho)protein profiles detected by flow cytometry analyses have been shown to correlate with ex vivo drug sensitivity and to predict treatment outcomes in hematologic cancers, demonstrating that this method is suitable for pre-clinical studies. Here, we present a flow cytometry protocol that combines multi-parameter immunophenotyping with single-cell (phospho)protein profiling. The protocol makes use of fluorescent cell barcoding, which means that multiple cell samples, either collected from different donors or exposed to different treatment conditions, can be combined and analyzed as one experiment. This reduces variability between samples, increases the throughput of the experiment, and lowers experimental costs. This protocol may serve as a guide for the use and further development of assays to study immunophenotype and cell signaling at single-cell resolution in normal and malignant cells. The read-outs may provide biological insight into cancer pathogenesis, identify novel drug targets, and ultimately serve as a biomarker to guide clinical decision-making., (© 2024. The Author(s).)

Increasing use of covalent and noncovalent inhibitors of Bruton's tyrosine kinase (BTK) has elucidated a series of acquired drug-resistant BTK mutations in patients with B cell malignancies. Here we identify inhibitor resistance mutations in BTK with distinct enzymatic activities, including some that impair BTK enzymatic activity while imparting novel protein-protein interactions that sustain B cell receptor (BCR) signaling. Furthermore, we describe a clinical-stage BTK and IKZF1/3 degrader, NX-2127, that can bind and proteasomally degrade each mutant BTK proteoform, resulting in potent blockade of BCR signaling. Treatment of chronic lymphocytic leukemia with NX-2127 achieves >80% degradation of BTK in patients and demonstrates proof-of-concept therapeutic benefit. These data reveal an oncogenic scaffold function of mutant BTK that confers resistance across clinically approved BTK inhibitors but is overcome by BTK degradation in patients.

Current treatment selection for acute myeloid leukemia (AML) patients depends on risk stratification based on cytogenetic and genomic markers. However, the forecasting accuracy of treatment response remains modest, with most patients receiving intensive chemotherapy. Recently, ex vivo drug screening has gained traction in personalized treatment selection and as a tool for mapping patient groups based on relevant cancer dependencies. Here, we systematically evaluated the use of drug sensitivity profiling for predicting patient survival and clinical response to chemotherapy in a cohort of AML patients. We compared computational methodologies for scoring drug efficacy and characterized tools to counter noise and batch-related confounders pervasive in high-throughput drug testing. We show that ex vivo drug sensitivity profiling is a robust and versatile approach to patient prognostics that comprehensively maps functional signatures of treatment response and disease progression. In conclusion, ex vivo drug profiling can assess risk for individual AML patients and may guide clinical decision-making., Competing Interests: Declaration of interests A.L. serves on the advisory board of Janssen, AbbVie, Servier, and Astellas and has received research funding from Gilead. S.S.S. has received honoraria from AbbVie and AstraZeneca and research support from BeiGene and TG Therapeutics., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (© 2023. The Author(s).)

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., (© 2023. The Author(s).)

Bruton tyrosine kinase (BTK) is essential for B-cell receptor (BCR) signaling, a driver of chronic lymphocytic leukemia (CLL). Covalent inhibitors bind C481 in the active site of BTK and have become a preferred CLL therapy. Disease progression on covalent BTK inhibitors is commonly associated with C481 mutations. Here, we investigated a targeted protein degrader, NRX-0492, that links a noncovalent BTK-binding domain to cereblon, an adaptor protein of the E3 ubiquitin ligase complex. NRX-0492 selectively catalyzes ubiquitylation and proteasomal degradation of BTK. In primary CLL cells, NRX-0492 induced rapid and sustained degradation of both wild-type and C481 mutant BTK at half maximal degradation concentration (DC50) of ≤0.2 nM and DC90 of ≤0.5 nM, respectively. Sustained degrader activity was maintained for at least 24 hours after washout and was equally observed in high-risk (deletion 17p) and standard-risk (deletion 13q only) CLL subtypes. In in vitro testing against treatment-naïve CLL samples, NRX-0492 was as effective as ibrutinib at inhibiting BCR-mediated signaling, transcriptional programs, and chemokine secretion. In patient-derived xenografts, orally administered NRX-0492 induced BTK degradation and inhibited activation and proliferation of CLL cells in blood and spleen and remained efficacious against primary C481S mutant CLL cells collected from a patient progressing on ibrutinib. Oral bioavailability, >90% degradation of BTK at subnanomolar concentrations, and sustained pharmacodynamic effects after drug clearance make this class of targeted protein degraders uniquely suitable for clinical translation, in particular as a strategy to overcome BTK inhibitor resistance. Clinical studies testing this approach have been initiated (NCT04830137, NCT05131022).

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Giliberto, Santana, Holien, Misund, Nakken, Vodak, Hovig, Meza-Zepeda, Coward, Waage, Taskén and Skånland.)

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., Competing Interests: Declaration of interests S.S.S. has received honoraria from AbbVie and AstraZeneca, and research support from BeiGene and TG Therapeutics. Å.F. has received honoraria from Bayer, Novartis, Pierre Fabre, Amgen, and Pfizer. The other authors declare no conflicts of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

As patients continue to live longer with chronic lymphocytic leukemia, it has become evident that there is an unmet treatment need for patients who have progressed on multiple lines of therapy. In this article, we attempt to define the "double refractory" patient as resistant to both Bruton's tyrosine kinase inhibitors (BTKi) and venetoclax for which prognosis is poor and there remains no standard of care. We further examine the mechanism of resistance to these targeted agents and discuss the current landscape for managing this patient population. Finally, we explore data supporting promising new agents, including non-covalent BTKi, chimeric antigen receptor T cells, and additional classes of agents currently in development., (© 2022 Wiley Periodicals LLC.)

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., (©2022 American Association for Cancer Research.)

Targeted therapies against phosphatidylinositol 3-kinase (PI3K), Bruton's tyrosine kinase (BTK), and B-cell lymphoma-2 (BCL-2) are approved for chronic lymphocytic leukemia (CLL). Since approval of the first-in-class drugs, next-generation agents have become available and are continuously under development. While these therapies act on well-characterized molecular targets, this knowledge is only to some extent taken into consideration when determining their dose in phase I trials. For example, BTK occupancy has been assessed in dose-finding studies of various BTK inhibitors, but the minimum doses that result in full BTK occupancy were not determined. Although targeted agents have a different dose-response relationship than cytotoxic agents, which are more effective near the maximum tolerated dose, the traditional 3 + 3 toxicity-driven trial design remains heavily used in the era of targeted therapies. If pharmacodynamic biomarkers were more stringently used to guide dose selection, the recommended phase II dose would likely be lower as compared to the toxicity-driven selection. Reduced drug doses may lower toxicity, which in some cases is severe for these agents, and are supported by retrospective studies demonstrating non-inferior outcomes for patients with clinically indicated dose reductions. Here, we review strategies that were used for dose selection in phase I studies of currently approved and select investigational targeted therapies in CLL, and discuss how our initial clinical experience with targeted therapies have pointed to dose reductions, intermittent dosing, and drug combinations as strategies to overcome treatment intolerance and resistance., (© 2022. The Author(s).)

Chronic lymphocytic leukaemia (CLL) is characterised by malignant mature-like B cells. Supportive to CLL cell survival is chronic B-cell receptor (BCR) signalling; however, emerging evidence demonstrates CLL cells proliferate in response to T-helper (Th) cells in a CD40L-dependent manner. We showed provision of Th stimulation via CD40L upregulated CD45 phosphatase activity and BCR signalling in non-malignant B cells. Consequently, we hypothesised Th cell upregulation of CLL cell CD45 activity may be an important regulator of CLL BCR signalling and proliferation. Using patient-derived CLL cells in a culture system with activated autologous Th cells, results revealed increases in both Th and CLL cell CD45 activity, which correlated with enhanced downstream antigen receptor signalling and proliferation. Concomitantly increased was the surface expression of Galectin-1, a CD45 ligand, and CD43, a CLL immunophenotypic marker. Galectin-1/CD43 double expression defined a proliferative CLL cell population with enhanced CD45 activity. Targeting either Galectin-1 or CD43 using silencing, pharmacology, or monoclonal antibody strategies dampened CD45 activity and CLL cell proliferation. These results highlight a mechanism where activated Th cells drive CLL cell BCR signalling and proliferation via Galectin-1 and CD43-mediated regulation of CD45 activity, identifying modulation of CD45 phosphatase activity as a potential therapeutic target in CLL., (© 2022 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

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., (© 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

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., (© 2022 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

In many complex diseases, such as cancers, resistance to monotherapies easily occurs, and longer-term treatment responses often require combinatorial therapies as next-line regimens. However, due to a massive number of possible drug combinations to test, there is a need for systematic and rational approaches to finding safe and effective drug combinations for each individual patient. This protocol describes an ecosystem of computational methods to guide high-throughput combinatorial screening that help experimental researchers to identify optimal drug combinations in terms of synergy, efficacy, and/or selectivity for further preclinical and clinical investigation. The methods are demonstrated in the context of combinatorial screening in primary cells of leukemia patients, where the translational aim is to identify drug combinations that show not only high synergy but also maximal cancer-selectivity. The mechanism-agnostic and cost-effective computational methods are widely applicable to various cancer types, which are amenable to drug testing, as the computational methods take as input only the phenotypic measurements of a subset of drug combinations, without requiring target information or genomic profiles of the patient samples., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Primary sclerosing cholangitis (PSC) is a chronic inflammatory liver disease without clear etiology or effective treatment. Genetic factors contribute to PSC pathogenesis, but so far, no causative mutation has been found. We performed whole-exome sequencing in a family with autosomal dominant inheritance of PSC and identified a heterozygous germline missense mutation in SEMA4D , encoding a K849T variant of CD100. The mutation was located in an evolutionarily conserved, unstructured cytosolic region of CD100 affecting downstream signaling. It was found to alter the function of CD100-expressing cells with a bias toward the T cell compartment that caused increased proliferation and impaired interferon-γ (IFN-γ) production after stimulation. Homologous mutation knock-in mice developed similar IFN-γ impairment in T cells and were more prone to develop severe cholangitis when exposed to 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet. Transfer of wild-type T cells to knock-in mice before and during DDC exposure attenuated cholangitis. Taken together, we identified an inherited mutation in the disordered cytosolic region of CD100 resulting in T cell functional defects. Our findings suggest a protective role for T cells in PSC that might be used therapeutically., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Insight into the critical role of B-cell receptor signaling for the pathogenesis of chronic lymphocytic leukemia (CLL) led to the development of targeted therapies directed at key regulators of cell survival. Agents targeting B-cell lymphoma-2 protein, Bruton's tyrosine kinase (BTK), and phosphatidylinositol 3-kinase are approved for treatment of CLL, and have significantly improved the disease management. Nevertheless, acquired resistance to the targeted therapies is a challenge still to be resolved. The mechanisms underlying resistance are becoming clearer, and include secondary mutations within the drug target and activation of bypass pathways. This knowledge has allowed development of strategies to prevent and overcome treatment resistance. Approaches to prevent resistance include targeting bypass mechanisms by combination therapies, temporally sequencing of therapies, improved clinical trial designs, and real-time monitoring of patient response. A rational design of drug sequencing may secure effective treatment options at the relapsed setting. Next-generation inhibitors and bispecific antibodies have the potential to overcome resistance to the BTK inhibitor ibrutinib. Immunotherapy, including chimeric antigen receptor-modified T-cell therapy, is explored for relapsed CLL. Here, recent advances that have contributed to the understanding of resistance to targeted therapies in CLL are discussed. Strategies for managing resistance are reviewed, including translational, real-world, and clinical perspectives., (© 2021 by The American Society of Hematology.)

The B cell receptor (BCR) is a master regulator of B cells, controlling cellular processes such as proliferation, migration and survival. Cell signalling downstream of the BCR is aberrantly activated in the B cell malignancy chronic lymphocytic leukaemia (CLL), supporting the pathophysiology of the disease. This insight has led to development and approval of small molecule inhibitors that target components of the BCR pathway. These advances have greatly improved the management of CLL, but the disease remains incurable. This may partly be explained by the inter-patient heterogeneity of the disease, also when it comes to treatment responses. Precision medicine is therefore required to optimize treatment and move towards a cure. Here, we discuss how the introduction of BCR signalling inhibitors has facilitated the development of functional in vitro assays to guide clinical treatment decisions on use of the same therapeutic agents in individual patients. The cellular responses to these agents can be analysed in high-throughput assays such as dynamic BH3 profiling, phospho flow experiments and drug sensitivity screens to identify predictive biomarkers. This progress exemplifies the positive synergy between basal and translational research needed to optimize patient care., (© 2019 The Authors. Scandinavian Journal of Immunology published by John Wiley & Sons Ltd on behalf of The Scandinavian Foundation for Immunology.)

Given advanced age, comorbidities, and immune dysfunction, chronic lymphocytic leukemia (CLL) patients may be at particularly high risk of infection and poor outcomes related to coronavirus disease 2019 (COVID-19). Robust analysis of outcomes for CLL patients, particularly examining effects of baseline characteristics and CLL-directed therapy, is critical to optimally manage CLL patients through this evolving pandemic. CLL patients diagnosed with symptomatic COVID-19 across 43 international centers (n = 198) were included. Hospital admission occurred in 90%. Median age at COVID-19 diagnosis was 70.5 years. Median Cumulative Illness Rating Scale score was 8 (range, 4-32). Thirty-nine percent were treatment naive ("watch and wait"), while 61% had received ≥1 CLL-directed therapy (median, 2; range, 1-8). Ninety patients (45%) were receiving active CLL therapy at COVID-19 diagnosis, most commonly Bruton tyrosine kinase inhibitors (BTKi's; n = 68/90 [76%]). At a median follow-up of 16 days, the overall case fatality rate was 33%, though 25% remain admitted. Watch-and-wait and treated cohorts had similar rates of admission (89% vs 90%), intensive care unit admission (35% vs 36%), intubation (33% vs 25%), and mortality (37% vs 32%). CLL-directed treatment with BTKi's at COVID-19 diagnosis did not impact survival (case fatality rate, 34% vs 35%), though the BTKi was held during the COVID-19 course for most patients. These data suggest that the subgroup of CLL patients admitted with COVID-19, regardless of disease phase or treatment status, are at high risk of death. Future epidemiologic studies are needed to assess severe acute respiratory syndrome coronavirus 2 infection risk, these data should be validated independently, and randomized studies of BTKi's in COVID-19 are needed to provide definitive evidence of benefit.

Recently, several small molecule drugs were approved for treatment of chronic lymphocytic leukemia (CLL), significantly improving patient management. However, knowledge about how to combine these therapies for optimal effects and what patients will best benefit from them is lacking. Here, we show that drug synergies can be identified by single cell signaling analyses. We investigated the effects of idelalisib, ibrutinib, and venetoclax on 35 protein epitopes by phospho flow in CLL cells. The activity of proteins in the B-cell receptor signalosome and the phosphatidylinositol 3-kinase pathway were altered upon drug exposure. Combined treatment with ibrutinib and venetoclax give promising results in clinical studies and we show that this combination exerted synergistic inhibitory effects on cell signaling and cell viability. Cell viability was monitored by flow cytometry and with independent drug sensitivity screens. Our analyses indicate that the standard dosages of ibrutinib and venetoclax can be lowered without loss of efficacy, potentially reducing drug costs, and toxicities. Observed correlation between signaling and viability indicates that signaling molecules could serve as biomarkers to predict response to therapy. We suggest that phospho flow should be considered as a novel approach for dose and synergy prediction in a precision medicine setting for CLL.

The prescription of drugs for depression is rising rapidly. One of the reasons for this trend is their many off-label uses. Up to one third of all prescriptions are for non-indicated use, which in addition to drug repurposing includes different dosing or duration than those recommended. In this review, we elaborate on what antidepressants can treat besides depression. The five classes of drugs for depression are introduced, and their mechanisms of action and serious side effects are described. The most common off-label uses of antidepressants are discussed, with a special focus on treating eating disorders, sleep problems, smoking cessation and managing chronic pain. Depression is often a comorbidity when antidepressants are chosen as therapy, but good therapeutic effects have been observed for other conditions also when depression is not involved. Finally, a new type of antidepressant developed from the hallucinogenic "party drug" ketamine is briefly introduced. This recent development suggests that antidepressants will keep playing a central role in medicine for years to come., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Full T cell activation depends on stimulation of the TCR in conjunction with a costimulatory receptor. The involvement of costimulatory molecules is potent, and a mechanistic understanding of how downstream signaling is regulated is required to fully understand T cell responsiveness. In this study, a proteomic approach was taken to identify the interactomes of the coreceptors CD2 and CD28. These coreceptors are both positive regulators of T cell activation, but CD28 less potently induces TCR-proximal signaling. C-terminal Src kinase (CSK), a negative regulator of TCR signaling, was identified as a specific and direct interactor only of activated CD28. CSK is recruited to CD28 upon T cell activation, and the in vitro kinase activity of CSK is enhanced in the presence of phosphorylated CD28. Interruption of the CSK/CD28 interaction prior to TCR/CD28 costimulation induces a signaling response which mimics the more potent CD2-induced TCR-proximal pathway activation. Thus, CD28 functions as a novel adaptor protein for CSK, and CSK regulates signaling downstream of CD28., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Phospho flow is a powerful approach to detect cell signaling aberrations, identify biomarkers and assess pharmacodynamics, and can be performed using cryopreserved samples. The effects of cryopreservation on signaling responses and the reproducibility of phospho flow measurements are however unknown in many cell systems. Here, B lymphocytes were isolated from healthy donors and patients with the B cell malignancy chronic lymphocytic leukemia and analyzed by phospho flow using phospho-specific antibodies targeting 20 different protein epitopes. Cells were analyzed both at basal conditions and after activation of cluster of differentiation 40 (CD40) or the B cell receptor. Pharmacodynamics of the novel pathway inhibitor ibrutinib was also assessed. At all conditions, fresh cells were compared to cryopreserved cells. Minimal variation between fresh and frozen samples was detected. Reproducibility was tested by running samples from the same donors in different experiments. The results demonstrate reproducibility across different phospho flow runs and support the use of cryopreserved samples in future phospho flow studies of B lymphocytes.

Aberrant cell signaling plays a central role in cancer development and progression. Most novel targeted therapies are indeed directed at proteins and protein functions, and cell signaling aberrations may therefore serve as biomarkers to indicate personalized treatment options. As opposed to DNA and RNA analyses, changes in protein activity can more efficiently evaluate the mechanisms underlying drug sensitivity and resistance. Phospho flow cytometry is a powerful technique that measures protein phosphorylation events at the cellular level, an important feature that distinguishes this method from other antibody-based approaches. The method allows for simultaneous analysis of multiple signaling proteins. In combination with fluorescent cell barcoding, larger medium- to high-throughput data-sets can be acquired by standard cytometer hardware in short time. Phospho flow cytometry has applications both in studies of basic biology and in clinical research, including signaling analysis, biomarker discovery and assessment of pharmacodynamics. Here, a detailed experimental protocol is provided for phospho flow analysis of purified peripheral blood mononuclear cells, using chronic lymphocytic leukemia cells as an example.

Chronic lymphocytic leukemia (CLL) has a high incidence and a steeply growing prevalence in the Western world. The heterogeneity of the disease necessitates individual mapping of biology and predicted drug response in each patient as basis for administration of tailored treatments. Cell signaling aberrations may serve as biological indicators for suitable therapy. By applying phospho-specific flow cytometry, we mapped basal and induced phosphorylation levels of 20 phospho-epitopes on proteins relevant to B-cell signaling in B cells from 22 CLL patients and 25 normal controls. The signaling response of the cytostatic drugs fludarabine, doxorubicin and vincristine was also investigated. CLL cells exerted similar or lower basal phosphorylation levels compared to normal B cells, with the exception of STAT3 (pY705) which was increased. Interestingly, STAT3 inhibitors normalized the STAT3 (pY705) level and reduced cell viability. Vincristine treatment significantly modulated phosphorylation levels in CLL cells, while no effect was observed in controls or after fludarabine or doxorubicin treatment. After BCR stimulation, CLL cells showed a tendency towards impaired phosphorylation levels, significant for several of the analyzed proteins. However, the level of Akt (pS473) was more potently induced in IgHV unmutated CLL (UM-CLL) patient samples and was significantly higher than in M-CLL samples. Importantly, the PI3Kδ inhibitor idelalisib potently reversed the effect of anti-IgM on Akt (pS473). Thus, signaling aberrations could be identified by phosphoflow cytometry and aberrant signaling could be normalized by small molecule drugs. This approach can identify relevant drug targets as well as drug effects in the individual patient., Competing Interests: CONFLICTS OF INTEREST The authors do not declare any conflicts of interest.

Full T-cell activation critically depends on the engagement of the TCR (T-cell receptor) in conjunction with a second signal by co-stimulatory receptors that boost the immune response. In the present study we have compared signalling patterns induced by the two co-receptors CD2 and CD28 in human peripheral blood T-cells. These co-receptors were previously suggested to be redundant in function. By a combination of multi-parameter phosphoflow cytometry, phosphokinase arrays and Western blot analyses, we demonstrate that CD2 co-stimulation induces phosphorylation of the TCR-proximal signalling complex, whereas CD28 activates distal signalling molecules, including the transcription factors NF-κB (nuclear factor κB), ATF (activating transcription factor)-2, STAT3/5 (signal transducer and activator of transcription 3/5), p53 and c-Jun. These signalling patterns were conserved in both naïve and effector/memory T-cell subsets. We show that free intracellular Ca(2+) and signalling through the PI3K (phosphoinositide 3-kinase)/Akt pathway are required for proper CD28-induced NF-κB activation. The signalling patterns induced by CD2 and CD28 co-stimulation lead to distinct functional immune responses in T-cell proliferation and cytokine production. In conclusion, CD2 and CD28 co-stimulation induces distinct signalling responses and functional outcomes in T-cells.

The AB plant toxin ricin binds both glycoproteins and glycolipids at the cell surface via its B subunit. After binding, ricin is endocytosed and then transported retrogradely through the Golgi to the endoplasmic reticulum (ER). In the ER, the A subunit is retrotranslocated to the cytosol in a chaperone-dependent process, which is not fully explored. Recently two separate siRNA screens have demonstrated that ER chaperones have implications for ricin toxicity. ER associated degradation (ERAD) involves translocation of misfolded proteins from ER to cytosol and it is conceivable that protein toxins exploit this pathway. The ER chaperone BiP is an important ER regulator and has been implicated in toxicity mediated by cholera and Shiga toxin. In this study, we have investigated the role of BiP in ricin translocation to the cytosol. We first show that overexpression of BiP inhibited ricin translocation and protected cells against the toxin. Furthermore, shRNA-mediated depletion of BiP enhanced toxin translocation resulting in increased cytotoxicity. BiP-dependent inhibition of ricin toxicity was independent of ER stress. Our findings suggest that in contrast to what was shown with the Shiga toxin, the presence of BiP does not facilitate, but rather inhibits the entry of ricin into the cytosol.

Objective: Interleukin (IL)-33 is a nuclear protein that is released from stressed or damaged cells to act as an alarmin. We investigated the effects of IL-33 on endothelial cells, using the prototype IL-1 family member, IL-1β, as a reference., Methods and Results: Human umbilical vein endothelial cells were stimulated with IL-33 or IL-1β, showing highly similar phosphorylation of signaling molecules, induction of adhesion molecules, and transcription profiles. However, intradermally injected IL-33 elicited significantly less proinflammatory endothelial activation when compared with IL-1β and led us to observe that quiescent endothelial cells (ppRb(low)p27(high)) were strikingly resistant to IL-33. Accordingly, the IL-33 receptor was preferentially expressed in nonquiescent cells of low-density cultures, corresponding to selective induction of adhesion molecules and chemokines. Multiparameter phosphoflow cytometry confirmed that signaling driven by IL-33 was stronger in nonquiescent cells. Manipulation of nuclear IL-33 expression by siRNA or adenoviral transduction revealed no functional link between nuclear, endogenous IL-33, and exogenous IL-33 responsiveness., Conclusions: In contrast to other inflammatory cytokines, IL-33 selectively targets nonquiescent endothelial cells. By this novel concept, quiescent cells may remain nonresponsive to a proinflammatory stimulus that concomitantly triggers a powerful response in cells that have been released from contact inhibition.

The transmembrane protein Cbp/PAG (Csk binding protein/phospho-protein associated with glycosphingolipid-enriched microdomains) has a negative regulatory role in T cell activation as an adapter for C-terminal Src kinase, Csk. In T cells, membrane docking of Csk is promoted by binding to FynT-phosphorylated Cbp/PAG (pTyr317) to allow targeting of substrates residing in lipid rafts. Here, we investigate a potential parallel position for Cbp/PAG and the Src kinase Lyn in early B cell receptor signaling. Using normal and transformed B cells, we have compared signal profiles of BCR-triggered responses created by phospho-specific flow cytometry. In human normal B cells, our data show that reduced Cbp/PAG levels leads to enhanced and prolonged activation of proximal signaling mediators, while over-expression of the adapter in normal, EBV-transformed cells results in reduced calcium flux. Taken together, our findings support a negative regulatory function for Cbp/PAG in proximal BCR signaling in these cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Annexins constitute a family of calcium and membrane binding proteins. As annexin A1 and A2 have previously been linked to various membrane trafficking events, we initiated this study to investigate the role of these annexins in the uptake and intracellular transport of the bacterial Shiga toxin (Stx) and the plant toxin ricin. Once endocytosed, both toxins are retrogradely transported from endosomes to the Golgi apparatus and the endoplasmic reticulum before being targeted to the cytosol where they inhibit protein synthesis. This study was performed to obtain new information both about toxin transport and the function of annexin A1 and annexin A2. Our data show that depletion of annexin A1 or A2 alters the retrograde transport of Stx but not ricin, without affecting toxin binding or internalization. Knockdown of annexin A1 increases Golgi transport of Stx, whereas knockdown of annexin A2 slightly decreases the same transport step. Interestingly, annexin A1 was found in proximity to cytoplasmic phospholipase A2 (cPLA(2)), and the basal as well as the increased Golgi transport of Stx upon annexin A1 knockdown is dependent on cPLA(2) activity. In conclusion, annexin A1 and A2 have different roles in Stx transport to the trans-Golgi network. The most prominent role is played by annexin A1 which normally works as a negative regulator of retrograde transport from the endosomes to the Golgi network, most likely by complex formation and inhibition of cPLA(2).

SHARPIN is a ubiquitin-binding and ubiquitin-like-domain-containing protein which, when mutated in mice, results in immune system disorders and multi-organ inflammation. Here we report that SHARPIN functions as a novel component of the linear ubiquitin chain assembly complex (LUBAC) and that the absence of SHARPIN causes dysregulation of NF-κB and apoptotic signalling pathways, explaining the severe phenotypes displayed by chronic proliferative dermatitis (cpdm) in SHARPIN-deficient mice. Upon binding to the LUBAC subunit HOIP (also known as RNF31), SHARPIN stimulates the formation of linear ubiquitin chains in vitro and in vivo. Coexpression of SHARPIN and HOIP promotes linear ubiquitination of NEMO (also known as IKBKG), an adaptor of the IκB kinases (IKKs) and subsequent activation of NF-κB signalling, whereas SHARPIN deficiency in mice causes an impaired activation of the IKK complex and NF-κB in B cells, macrophages and mouse embryonic fibroblasts (MEFs). This effect is further enhanced upon concurrent downregulation of HOIL-1L (also known as RBCK1), another HOIP-binding component of LUBAC. In addition, SHARPIN deficiency leads to rapid cell death upon tumour-necrosis factor α (TNF-α) stimulation via FADD- and caspase-8-dependent pathways. SHARPIN thus activates NF-κB and inhibits apoptosis via distinct pathways in vivo.

The spatial organization of transmembrane receptors is a critical step in signal transduction and receptor trafficking in cells. Transmembrane receptors engage in lateral homotypic and heterotypic cis-interactions as well as intercellular trans-interactions that result in the formation of signalling foci for the initiation of different signalling networks. Several aspects of ligand-induced receptor clustering and association with signalling proteins are also influenced by the lipid composition of membranes. Thus, lipid microdomains have a function in tuning the activity of many transmembrane receptors by positively or negatively affecting receptor clustering and signal transduction. We review the current knowledge about the functions of clustering of transmembrane receptors and lipid-protein interactions important for the spatial organization of signalling at the membrane.

Shiga toxin (Stx) is a bacterial toxin that binds to its receptor Gb3 at the plasma membrane. It is taken up by endocytosis and transported retrogradely via the Golgi apparatus to the endoplasmic reticulum. The toxin is then translocated to the cytosol where it exerts its toxic effect. We have previously shown that phosphorylation of clathrin heavy chain (CHC) is an early event following Stx binding to HeLa cells, and that this requires the activity of the tyrosine kinase Syk. Here, we have investigated this event in more detail in the B lymphoid cell line Ramos, which expresses high endogenous levels of both Syk and Gb3. We report that efficient endocytosis of Stx in Ramos cells requires Syk activity and that Syk is recruited to the uptake site of Stx. Furthermore, in response to Stx treatment, CHC and Syk were rapidly phosphorylated in a Src family kinase dependent manner at Y1477 and Y352, respectively. We show that these phosphorylated residues act as binding sites for the direct interaction between Syk and CHC. Interestingly, Syk-CHC complex formation could be induced by both Stx and B cell receptor stimulation.

Background: Sorting nexins (SNXs) constitute a family of proteins classified by their phosphatidylinositol (PI) binding Phox homology (PX) domain. Some members regulate intracellular trafficking. We have here investigated mechanisms underlying SNX4 mediated endosome to Golgi transport., Methodology/principal Findings: We show that SNX4 forms complexes with clathrin and dynein. The interactions were inhibited by wortmannin, a PI3-kinase inhibitor, suggesting that they form when SNX4 is associated with PI(3)P on endosomes. We further localized the clathrin interacting site on SNX4 to a clathrin box variant. A short peptide containing this motif was sufficient to pull down both clathrin and dynein. Knockdown studies demonstrated that clathrin is not required for the SNX4/dynein interaction. Moreover, clathrin knockdown led to increased Golgi transport of the toxin ricin, as well as redistribution of endosomes., Conclusions/significance: We discuss the possibility of clathrin serving as a regulator of SNX4-dependent transport. Upon clathrin release, dynein may bind SNX4 and mediate retrograde movement.

Shiga toxin (Stx) is after endocytosis transported via early endosomes to the Golgi apparatus and endoplasmic reticulum. It is then translocated to the cytosol where it exerts its toxic effect. We recently reported that p38 is required for endosome to Golgi transport of Stx. In the present study, we investigated whether β-arrestins are effectors of this pathway. β-arrestin knockdown led to enhanced Stx transport. A similar phenotype was achieved upon p38 activation. We demonstrate that p38 and β-arrestin act on the same pathway. β-arrestin colocalized with internalized Stx and, interestingly, was recruited to endosomes upon p38 activation. After Stx treatment, p38 and β-arrestin formed a transient complex. From these data we propose that β-arrestin negatively regulates Stx transport via an interaction with activated p38 and attenuation of its signalling. Interestingly, also mannose 6-phosphate receptor transport was regulated by p38 and β-arrestin. β-arrestins therefore seem to regulate an endosome to Golgi pathway used by multiple cargo proteins., (© 2009 Blackwell Publishing Ltd.)

Shiga toxin (Stx) binds to the cell, and it is transported via endosomes and the Golgi apparatus to the endoplasmic reticulum and cytosol, where it exerts its toxic effect. We have recently shown that Stx activates the tyrosine kinase Syk, which in turn induces clathrin phosphorylation and up-regulates Stx uptake. Here, we show that toxin-induced signaling can also regulate another step in intracellular Stx transport. We demonstrate that transport of Stx to the Golgi apparatus is dependent on the mitogen-activated protein kinase p38. Treatment of cells with chemical inhibitors or small interfering RNA targeting p38 inhibited Stx transport to the Golgi and reduced Stx toxicity. This p38 dependence is specific to Stx, because transport of the related toxin ricin was not affected by p38 inhibition. Stx rapidly activated p38, and recruited it to early endosomes in a Ca(2+)-dependent manner. Furthermore, agonist-induced oscillations in cytosolic Ca(2+) levels were inhibited upon Stx stimulation, possibly reflecting Stx-dependent local alterations in cytosolic Ca(2+) levels. Intracellular transport of Stx is Ca(2+) dependent, and we provide evidence that Stx activates a signaling cascade involving cross talk between Ca(2+) and p38, to regulate its trafficking to the Golgi apparatus.

The SecY complex associates with the ribosome to form a protein translocation channel in the bacterial plasma membrane. We have used cryo-electron microscopy and quantitative mass spectrometry to show that a nontranslating E. coli ribosome binds to a single SecY complex. The crystal structure of an archaeal SecY complex was then docked into the electron density maps. In the resulting model, two cytoplasmic loops of SecY extend into the exit tunnel near proteins L23, L29, and L24. The loop between transmembrane helices 8 and 9 interacts with helices H59 and H50 in the large subunit RNA, while the 6/7 loop interacts with H7. We also show that point mutations of basic residues within either loop abolish ribosome binding. We suggest that SecY binds to this primary site on the ribosome and subsequently captures and translocates the nascent chain.

The bacterial toxin Shiga toxin (Stx) is transported retrogradely from early endosomes to the Golgi apparatus on its way to the endoplasmic reticulum (ER) and the cytosol. In this study we explored the functions of the two phosphoinositide binding proteins Sorting nexin 1 (SNX1) and Sorting nexin 2 (SNX2) in endosomal sorting of the toxin. When Vero cells were depleted of either SNX1 or SNX2 by small interfering RNA (siRNA), Stx transport to the trans-Golgi network (TGN) was impaired by > or = 40%, whereas combined depletion of SNX1 and SNX2 gave a total inhibition of approximately 80%. Inhibition of PI(3)P formation by wortmannin resulted in a similar reduction. Thus, although being partly redundant, both SNX1 and SNX2 are required for efficient Stx trafficking to the Golgi apparatus.

Protein kinase C (PKC) isozymes regulate different vesicular trafficking steps in the recycling or degradative pathways. However, a possible role of these kinases in the retrograde pathway from endosomes to the Golgi complex has previously not been investigated. We report here the involvement of a specific PKC isozyme, PKCdelta, in the intracellular transport of the glycolipid-binding Shiga toxin (Stx), which utilizes the retrograde pathway to intoxicate cells. Upon binding to cells, Stx was shown to specifically activate PKCdelta and not PKCalpha. The involvement of PKCdelta and PKCalpha in the retrograde transport of Stx was then monitored biochemically and by immunofluorescence after inhibition or depletion of the isozymes. PKCdelta, but not PKCalpha, was shown to selectively regulate the endosome-to-Golgi transport of StxB. Upon inhibition or knockdown of PKCdelta, StxB molecules colocalized less with giantin and more with EEA1, indicating that the molecules were accumulated in endosomes, unable to reach the Golgi complex. The inhibition of Golgi transport of Stx was reflected by a strong reduction in the toxic effect, demonstrating that transport of Stx to the cytosol is dependent on PKCdelta activity. These results are in agreement with our previous data, which show that Stx is able to stimulate its own transport.

The plant toxin ricin is transported from the plasma membrane via early endosomes and the Golgi apparatus to the endoplasmic reticulum. From this compartment, it enters the cytosol and inhibits protein synthesis. Lipid phosphorylation is an important regulator of vesicular transport, and in the present study we have investigated the role of the phosphatidylinositol (PI) 3-kinase hVps34 in retrograde transport of ricin. Our data demonstrate that transport of ricin from endosomes to the Golgi apparatus in human embryonic kidney cells (HEK 293) is dependent on PI(3)P. By using PI 3-kinase inhibitors, by sequestering the hVps34 product PI(3)P and by expressing mutants of hVps34 or small interfering RNA targeted against its messenger RNA, we show that hVps34 and its product PI(3)P are involved in transport of ricin from endosome to Golgi apparatus. Furthermore, we identify two effector proteins in the hVps34-dependent pathway, namely sorting nexin (SNX) 2 and SNX4. Knockdown of SNX2 or SNX4 inhibits ricin transport to the Golgi apparatus to the same extent as when hVps34 is perturbed. Furthermore, inhibition or knockdown of hVps34 redistributes these proteins. Interestingly, knocking down both SNX2 and SNX4 results in a better inhibition than knocking down only one of them, suggesting that they may act on separate pathways.