Mucus sialylation determines intestinal host-commensal homeostasis.

Intestinal mucus forms the first line of defense against bacterial invasion while providing nutrition to support microbial symbiosis. How the host controls mucus barrier integrity and commensalism is unclear. We show that terminal sialylation of glycans on intestinal mucus by ST6GALNAC1 (ST6), the dominant sialyltransferase specifically expressed in goblet cells and induced by microbial pathogen-associated molecular patterns, is essential for mucus integrity and protecting against excessive bacterial proteolytic degradation. Glycoproteomic profiling and biochemical analysis of ST6 mutations identified in patients show that decreased sialylation causes defective mucus proteins and congenital inflammatory bowel disease (IBD). Mice harboring a patient ST6 mutation have compromised mucus barriers, dysbiosis, and susceptibility to intestinal inflammation. Based on our understanding of the ST6 regulatory network, we show that treatment with sialylated mucin or a Foxo3 inhibitor can ameliorate IBD.

Sequential inverse dysregulation of the RNA helicases DDX3X and DDX3Y facilitates MYC-driven lymphomagenesis.

DDX3X is a ubiquitously expressed RNA helicase involved in multiple stages of RNA biogenesis. DDX3X is frequently mutated in Burkitt lymphoma, but the functional basis for this is unknown. Here, we show that loss-of-function DDX3X mutations are also enriched in MYC-translocated diffuse large B cell lymphoma and reveal functional cooperation between mutant DDX3X and MYC. DDX3X promotes the translation of mRNA encoding components of the core translational machinery, thereby driving global protein synthesis. Loss-of-function DDX3X mutations moderate MYC-driven global protein synthesis, thereby buffering MYC-induced proteotoxic stress during early lymphomagenesis. Established lymphoma cells restore full protein synthetic capacity by aberrant expression of DDX3Y, a Y chromosome homolog, the expression of which is normally restricted to the testis. These findings show that DDX3X loss of function can buffer MYC-driven proteotoxic stress and highlight the capacity of male B cell lymphomas to then compensate for this loss by ectopic DDX3Y expression.

Epigenetic control over the cell-intrinsic immune response antagonizes self-renewal in acute myeloid leukemia.

ABSTRACT: Epigenetic modulation of the cell-intrinsic immune response holds promise as a therapeutic approach for leukemia. However, current strategies designed for transcriptional activation of endogenous transposons and subsequent interferon type-I (IFN-I) response, show limited clinical efficacy. Histone lysine methylation is an epigenetic signature in IFN-I response associated with suppression of IFN-I and IFN-stimulated genes, suggesting histone demethylation as key mechanism of reactivation. In this study, we unveil the histone demethylase PHF8 as a direct initiator and regulator of cell-intrinsic immune response in acute myeloid leukemia (AML). Site-specific phosphorylation of PHF8 orchestrates epigenetic changes that upregulate cytosolic RNA sensors, particularly the TRIM25-RIG-I-IFIT5 axis, thereby triggering the cellular IFN-I response-differentiation-apoptosis network. This signaling cascade largely counteracts differentiation block and growth of human AML cells across various disease subtypes in vitro and in vivo. Through proteome analysis of over 200 primary AML bone marrow samples, we identify a distinct PHF8/IFN-I signature in half of the patient population, without significant associations with known clinically or genetically defined AML subgroups. This profile was absent in healthy CD34+ hematopoietic progenitor cells, suggesting therapeutic applicability in a large fraction of patients with AML. Pharmacological support of PHF8 phosphorylation significantly impairs the growth in samples from patients with primary AML. These findings provide novel opportunities for harnessing the cell-intrinsic immune response in the development of immunotherapeutic strategies against AML.

Actinin-4 controls survival signaling in B cells by limiting the lateral mobility of B-cell antigen receptors.

The structure and dynamics of F-actin networks in the cortical area of B cells control the signal efficiency of B-cell antigen receptors (BCRs). Although antigen-induced signaling has been studied extensively, the role of cortical F-actin in antigen-independent tonic BCR signaling is less well understood. Because these signals are essential for the survival of B cells and are consequently exploited by several B-cell lymphomas, we assessed how the cortical F-actin structure influences tonic BCR signal transduction. We employed genetic variants of a primary cell-like B-cell line that can be rendered quiescent to show that cross-linking of actin filaments by α-actinin-4 (ACTN4), but not ACTN1, is required to preserve the dense architecture of F-actin in the cortical area of B cells. The reduced cortical F-actin density in the absence of ACTN4 resulted in increased lateral BCR diffusion. Surprisingly, this was associated with reduced tonic activation of BCR-proximal effector proteins, extracellular signal-regulated kinase, and pro-survival pathways. Accordingly, ACTN4-deficient B-cell lines and primary human B cells exhibit augmented apoptosis. Hence, our findings reveal that cortical F-actin architecture regulates antigen-independent tonic BCR survival signals in human B cells.

Nck-mediated recruitment of BCAP to the BCR regulates the PI(3)K-Akt pathway in B cells.

The adaptor Nck links receptor signaling to cytoskeleton regulation. Here we found that Nck also controlled the phosphatidylinositol-3-OH kinase (PI(3)K)-kinase Akt pathway by recruiting the adaptor BCAP after activation of B cells. Nck bound directly to the B cell antigen receptor (BCR) via the non-immunoreceptor tyrosine-based activation motif (ITAM) phosphorylated tyrosine residue at position 204 in the tail of the immunoglobulin-α component. Genetic ablation of Nck resulted in defective BCR signaling, which led to hampered survival and proliferation of B cells in vivo. Indeed, antibody responses in Nck-deficient mice were also considerably impaired. Thus, we demonstrate a previously unknown adaptor function for Nck in recruiting BCAP to sites of BCR signaling and thereby modulating the PI(3)K-Akt pathway in B cells.

Early B cell factor 4 modulates FAS-mediated apoptosis and promotes cytotoxic function in human immune cells.

Apoptosis is a genetically regulated program of cell death that plays a key role in immune disease processes. We identified EBF4, a little-studied member of the early B cell factor (EBF) family of transcription factors, in a whole-genome CRISPR screen for regulators of Fas/APO-1/CD95-mediated T cell death. Loss of EBF4 increases the half-life of the c-FLIP protein, and its presence in the Fas signaling complex impairs caspase-8 cleavage and apoptosis. Transcriptome analysis revealed that EBF4 regulates molecules such as TBX21, EOMES, granzyme, and perforin that are important for human natural killer (NK) and CD8+ T cell functions. Proximity-dependent biotin identification (Bio-ID) mass spectrometry analyses showed EBF4 binding to STAT3, STAT5, and MAP kinase 3 and a strong pathway relationship to interleukin-2 regulated genes, which are known to govern cytotoxicity pathways. Chromatin immunoprecipitation and DNA sequencing analysis defined a canonical EBF4 binding motif, 5'-CCCNNGG/AG-3', closely related to the EBF1 binding site; using a luciferase-based reporter, we found a dose-dependent transcriptional response of this motif to EBF4. We also conducted assay for transposase-accessible chromatin sequencing in EBF4-overexpressing cells and found increased chromatin accessibility upstream of granzyme and perforin and in topologically associated domains in human lymphocytes. Finally, we discovered that the EBF4 has basal expression in human but not mouse NK cells and CD8+ T cells and vanishes following activating stimulation. Together, our data reveal key features of a previously unknown transcriptional regulator of human cytotoxic immune function.

SHMT2 inhibition disrupts the TCF3 transcriptional survival program in Burkitt lymphoma.

Burkitt lymphoma (BL) is an aggressive lymphoma type that is currently treated by intensive chemoimmunotherapy. Despite the favorable clinical outcome for most patients with BL, chemotherapy-related toxicity and disease relapse remain major clinical challenges, emphasizing the need for innovative therapies. Using genome-scale CRISPR-Cas9 screens, we identified B-cell receptor (BCR) signaling, specific transcriptional regulators, and one-carbon metabolism as vulnerabilities in BL. We focused on serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in one-carbon metabolism. Inhibition of SHMT2 by either knockdown or pharmacological compounds induced anti-BL effects in vitro and in vivo. Mechanistically, SHMT2 inhibition led to a significant reduction of intracellular glycine and formate levels, which inhibited the mTOR pathway and thereby triggered autophagic degradation of the oncogenic transcription factor TCF3. Consequently, this led to a collapse of tonic BCR signaling, which is controlled by TCF3 and is essential for BL cell survival. In terms of clinical translation, we also identified drugs such as methotrexate that synergized with SHMT inhibitors. Overall, our study has uncovered the dependency landscape in BL, identified and validated SHMT2 as a drug target, and revealed a mechanistic link between SHMT2 and the transcriptional master regulator TCF3, opening up new perspectives for innovative therapies.

A multiprotein supercomplex controlling oncogenic signalling in lymphoma.

B cell receptor (BCR) signalling has emerged as a therapeutic target in B cell lymphomas, but inhibiting this pathway in diffuse large B cell lymphoma (DLBCL) has benefited only a subset of patients1. Gene expression profiling identified two major subtypes of DLBCL, known as germinal centre B cell-like and activated B cell-like (ABC)2,3, that show poor outcomes after immunochemotherapy in ABC. Autoantigens drive BCR-dependent activation of NF-κB in ABC DLBCL through a kinase signalling cascade of SYK, BTK and PKCß to promote the assembly of the CARD11-BCL10-MALT1 adaptor complex, which recruits and activates IκB kinase4-6. Genome sequencing revealed gain-of-function mutations that target the CD79A and CD79B BCR subunits and the Toll-like receptor signalling adaptor MYD885,7, with MYD88(L265P) being the most prevalent isoform. In a clinical trial, the BTK inhibitor ibrutinib produced responses in 37% of cases of ABC1. The most striking response rate (80%) was observed in tumours with both CD79B and MYD88(L265P) mutations, but how these mutations cooperate to promote dependence on BCR signalling remains unclear. Here we used genome-wide CRISPR-Cas9 screening and functional proteomics to determine the molecular basis of exceptional clinical responses to ibrutinib. We discovered a new mode of oncogenic BCR signalling in ibrutinib-responsive cell lines and biopsies, coordinated by a multiprotein supercomplex formed by MYD88, TLR9 and the BCR (hereafter termed the My-T-BCR supercomplex). The My-T-BCR supercomplex co-localizes with mTOR on endolysosomes, where it drives pro-survival NF-κB and mTOR signalling. Inhibitors of BCR and mTOR signalling cooperatively decreased the formation and function of the My-T-BCR supercomplex, providing mechanistic insight into their synergistic toxicity for My-T-BCR+ DLBCL cells. My-T-BCR supercomplexes characterized ibrutinib-responsive malignancies and distinguished ibrutinib responders from non-responders. Our data provide a framework for the rational design of oncogenic signalling inhibitors in molecularly defined subsets of DLBCL.

SH2 domain-containing inositol 5-phosphatases support the survival of Burkitt lymphoma cells by promoting energy metabolism.

Burkitt lymphoma cells (BL) exploit antigen-independent tonic signals transduced by the B cell antigen receptor (BCR) for their survival, but the molecular details of the rewired BLspecific BCR signal network remain unclear. A loss of function screen revealed the SH2 domain-containing 5`-inositol phosphatase 2 (SHIP2) as a potential modulator of BL fitness. We characterized the role of SHIP2 in BL survival in several BL cell models and show that perturbing SHIP2 function renders cells more susceptible to apoptosis, while attenuating proliferation in a BCR-dependent manner. Unexpectedly, SHIP2 deficiency did neither affect PI3K survival signals nor MAPK activity, but attenuated ATP production. We found that an efficient energy metabolism in BL cells requires phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2), which is the enzymatic product of SHIP proteins. Consistently, interference with the function of SHIP1 and SHIP2 augments BL cell susceptibility to PI3K inhibition. Notably, we here provide a molecular basis of how tonic BCR signals are connected to energy supply, which is particularly important for such an aggressively growing neoplasia. These findings may help to improve therapies for the treatment of BL by limiting energy metabolism through the inhibition of SHIP proteins, which renders BL cells more susceptible to the targeting of survival signals.

Profile of the multicenter cohort of the German Cancer Consortium's Clinical Communication Platform.

Treatment concepts in oncology are becoming increasingly personalized and diverse. Successively, changes in standards of care mandate continuous monitoring of patient pathways and clinical outcomes based on large, representative real-world data. The German Cancer Consortium's (DKTK) Clinical Communication Platform (CCP) provides such opportunity. Connecting fourteen university hospital-based cancer centers, the CCP relies on a federated IT-infrastructure sourcing data from facility-based cancer registry units and biobanks. Federated analyses resulted in a cohort of 600,915 patients, out of which 232,991 were incident since 2013 and for which a comprehensive documentation is available. Next to demographic data (i.e., age at diagnosis: 2.0% 0-20 years, 8.3% 21-40 years, 30.9% 41-60 years, 50.1% 61-80 years, 8.8% 81+ years; and gender: 45.2% female, 54.7% male, 0.1% other) and diagnoses (five most frequent tumor origins: 22,523 prostate, 18,409 breast, 15,575 lung, 13,964 skin/malignant melanoma, 9005 brain), the cohort dataset contains information about therapeutic interventions and response assessments and is connected to 287,883 liquid and tissue biosamples. Focusing on diagnoses and therapy-sequences, showcase analyses of diagnosis-specific sub-cohorts (pancreas, larynx, kidney, thyroid gland) demonstrate the analytical opportunities offered by the cohort's data. Due to its data granularity and size, the cohort is a potential catalyst for translational cancer research. It provides rapid access to comprehensive patient groups and may improve the understanding of the clinical course of various (even rare) malignancies. Therefore, the cohort may serve as a decisions-making tool for clinical trial design and contributes to the evaluation of scientific findings under real-world conditions.

The non-apoptotic function of Caspase-8 in negatively regulating the CDK9-mediated Ser2 phosphorylation of RNA polymerase II in cervical cancer.

Cervical cancer is the fourth most frequently diagnosed and fatal gynecological cancer. 15-61% of all cases metastasize and develop chemoresistance, reducing the 5-year survival of cervical cancer patients to as low as 17%. Therefore, unraveling the mechanisms contributing to metastasis is critical in developing better-targeted therapies against it. Here, we have identified a novel mechanism where nuclear Caspase-8 directly interacts with and inhibits the activity of CDK9, thereby modulating RNAPII-mediated global transcription, including those of cell-migration- and cell-invasion-associated genes. Crucially, low Caspase-8 expression in cervical cancer patients leads to poor prognosis, higher CDK9 phosphorylation at Thr186, and increased RNAPII activity in cervical cancer cell lines and patient biopsies. Caspase-8 knock-out cells were also more resistant to the small-molecule CDK9 inhibitor BAY1251152 in both 2D- and 3D-culture conditions. Combining BAY1251152 with Cisplatin synergistically overcame chemoresistance of Caspase-8-deficient cervical cancer cells. Therefore, Caspase-8 expression could be a marker in chemoresistant cervical tumors, suggesting CDK9 inhibitor treatment for their sensitization to Cisplatin-based chemotherapy.

Regulation of B cell receptor-dependent NF-κB signaling by the tumor suppressor KLHL14.

The KLHL14 gene acquires frequent inactivating mutations in mature B cell malignancies, especially in the MYD88L265P, CD79B mutant (MCD) genetic subtype of diffuse large B cell lymphoma (DLBCL), which relies on B cell receptor (BCR) signaling for survival. However, the pathogenic role of KLHL14 in DLBCL and its molecular function are largely unknown. Here, we report that KLHL14 is in close proximity to the BCR in the endoplasmic reticulum of MCD cell line models and promotes the turnover of immature glycoforms of BCR subunits, reducing total cellular BCR levels. Loss of KLHL14 confers relative resistance to the Bruton tyrosine kinase (BTK) inhibitor ibrutinib and promotes assembly of the MYD88-TLR9-BCR (My-T-BCR) supercomplex, which initiates prosurvival NF-κB activation. Consequently, KLHL14 inactivation allows MCD cells to maintain NF-κB signaling in the presence of ibrutinib. These findings reinforce the central role of My-T-BCR-dependent NF-κB signaling in MCD DLBCL and suggest that the genetic status of KLHL14 should be considered in clinical trials testing inhibitors of BTK and BCR signaling mediators in DLBCL.

Rewiring of B cell receptor signaling by Epstein-Barr virus LMP2A.

Epstein-Barr virus (EBV) infects human B cells and reprograms them to allow virus replication and persistence. One key viral factor in this process is latent membrane protein 2A (LMP2A), which has been described as a B cell receptor (BCR) mimic promoting malignant transformation. However, how LMP2A signaling contributes to tumorigenesis remains elusive. By comparing LMP2A and BCR signaling in primary human B cells using phosphoproteomics and transcriptome profiling, we identified molecular mechanisms through which LMP2A affects B cell biology. Consistent with the literature, we found that LMP2A mimics a subset of BCR signaling events, including tyrosine phosphorylation of the kinase SYK, the calcium initiation complex consisting of BLNK, BTK, and PLCγ2, and its downstream transcription factor NFAT. However, the majority of LMP2A-induced signaling events markedly differed from those induced by BCR stimulation. These included differential phosphorylation of kinases, phosphatases, adaptor proteins, transcription factors such as nuclear factor κB (NF-κB) and TCF3, as well as widespread changes in the transcriptional output of LMP2A-expressing B cells. LMP2A affected apoptosis and cell-cycle checkpoints by dysregulating the expression of apoptosis regulators such as BCl-xL and the tumor suppressor retinoblastoma-associated protein 1 (RB1). LMP2A cooperated with MYC and mutant cyclin D3, two oncogenic drivers of Burkitt lymphoma, to promote proliferation and survival of primary human B cells by counteracting MYC-induced apoptosis and by inhibiting RB1 function, thereby promoting cell-cycle progression. Our results indicate that LMP2A is not a pure BCR mimic but rather rewires intracellular signaling in EBV-infected B cells that optimizes cell survival and proliferation, setting the stage for oncogenic transformation.

T-cell-derived Hodgkin lymphoma has motility characteristics intermediate between Hodgkin and anaplastic large cell lymphoma.

Classic Hodgkin lymphoma (cHL) is usually characterized by a low tumour cell content, derived from crippled germinal centre B cells. Rare cases have been described in which the tumour cells show clonal T-cell receptor rearrangements. From a clinicopathological perspective, it is unclear if these cases should be classified as cHL or anaplastic large T-cell lymphoma (ALCL). Since we recently observed differences in the motility of ALCL and cHL tumour cells, here, we aimed to obtain a better understanding of T-cell-derived cHL by investigating their global proteomic profiles and their motility. In a proteomics analysis, when only motility-associated proteins were regarded, T-cell-derived cHL cell lines showed the highest similarity to ALK- ALCL cell lines. In contrast, T-cell-derived cHL cell lines presented a very low overall motility, similar to that observed in conventional cHL. Whereas all ALCL cell lines, as well as T-cell-derived cHL, predominantly presented an amoeboid migration pattern with uropod at the rear, conventional cHL never presented with uropods. The migration of ALCL cell lines was strongly impaired upon application of different inhibitors. This effect was less pronounced in cHL cell lines and almost invisible in T-cell-derived cHL. In summary, our cell line-derived data suggest that based on proteomics and migration behaviour, T-cell-derived cHL is a neoplasm that shares features with both cHL and ALCL and is not an ALCL with low tumour cell content. Complementary clinical studies on this lymphoma are warranted.

Strategies for Proteome-Wide Quantification of Glycosylation Macro- and Micro-Heterogeneity.

Protein glycosylation governs key physiological and pathological processes in human cells. Aberrant glycosylation is thus closely associated with disease progression. Mass spectrometry (MS)-based glycoproteomics has emerged as an indispensable tool for investigating glycosylation changes in biological samples with high sensitivity. Following rapid improvements in methodologies for reliable intact glycopeptide identification, site-specific quantification of glycopeptide macro- and micro-heterogeneity at the proteome scale has become an urgent need for exploring glycosylation regulations. Here, we summarize recent advances in N- and O-linked glycoproteomic quantification strategies and discuss their limitations. We further describe a strategy to propagate MS data for multilayered glycopeptide quantification, enabling a more comprehensive examination of global and site-specific glycosylation changes. Altogether, we show how quantitative glycoproteomics methods explore glycosylation regulation in human diseases and promote the discovery of biomarkers and therapeutic targets.

Evaluation and Optimization of High-Field Asymmetric Waveform Ion-Mobility Spectrometry for Multiplexed Quantitative Site-Specific N-Glycoproteomics.

The heterogeneity and complexity of glycosylation hinder the depth of site-specific glycoproteomics analysis. High-field asymmetric-waveform ion-mobility spectrometry (FAIMS) has been shown to improve the scope of bottom-up proteomics. The benefits of FAIMS for quantitative N-glycoproteomics have not been investigated yet. In this work, we optimized FAIMS settings for N-glycopeptide identification, with or without the tandem mass tag (TMT) label. The optimized FAIMS approach significantly increased the identification of site-specific N-glycopeptides derived from the purified immunoglobulin M (IgM) protein or human lymphoma cells. We explored in detail the changes in FAIMS mobility caused by N-glycopeptides with different characteristics, including TMT labeling, charge state, glycan type, peptide sequence, glycan size, and precursor m/z. Importantly, FAIMS also improved multiplexed N-glycopeptide quantification, both with the standard MS2 acquisition method and with our recently developed Glyco-SPS-MS3 method. The combination of FAIMS and Glyco-SPS-MS3 methods provided the highest quantitative accuracy and precision. Our results demonstrate the advantages of FAIMS for improved mass spectrometry-based qualitative and quantitative N-glycoproteomics.

Loss of the selective autophagy receptor p62 impairs murine myeloid leukemia progression and mitophagy.

Autophagy maintains hematopoietic stem cell integrity and prevents malignant transformation. In addition to bulk degradation, selective autophagy serves as an intracellular quality control mechanism and requires autophagy receptors, such as p62 (SQSTM1), to specifically bridge the ubiquitinated cargos into autophagosomes. Here, we investigated the function of p62 in acute myeloid leukemia (AML) in vitro and in murine in vivo models of AML. Loss of p62 impaired expansion and colony-forming ability of leukemia cells and prolonged latency of leukemia development in mice. High p62 expression was associated with poor prognosis in human AML. Using quantitative mass spectrometry, we identified enrichment of mitochondrial proteins upon immunoprecipitation of p62. Loss of p62 significantly delayed removal of dysfunctional mitochondria, increased mitochondrial superoxide levels, and impaired mitochondrial respiration. Moreover, we demonstrated that the autophagy-dependent function of p62 is essential for cell growth and effective mitochondrial degradation by mitophagy. Our results highlight the prominent role of selective autophagy in leukemia progression, and specifically, the importance of mitophagy to maintain mitochondrial integrity.

The vascular bone marrow niche influences outcome in chronic myeloid leukemia via the E-selectin - SCL/TAL1 - CD44 axis.

The endosteal bone marrow niche and vascular endothelial cells provide sanctuaries for leukemic cells. In murine chronic myeloid leukemia (CML) CD44 on leukemia cells and E-selectin on bone marrow endothelium are essential mediators for the engraftment of leukemic stem cells. We hypothesized that non-adhesion of CML-initiating cells to E-selectin on the bone marrow endothelium may lead to superior eradication of leukemic stem cells in CML after treatment with imatinib than imatinib alone. Indeed, here we show that treatment with the E-selectin inhibitor GMI-1271 in combination with imatinib prolongs survival of mice with CML via decreased contact time of leukemia cells with bone marrow endothelium. Non-adhesion of BCR-ABL1+ cells leads to an increase of cell cycle progression and an increase of expression of the hematopoietic transcription factor and proto-oncogene Scl/Tal1 in leukemia-initiating cells. We implicate SCL/TAL1 as an indirect phosphorylation target of BCR-ABL1 and as a negative transcriptional regulator of CD44 expression. We show that increased SCL/TAL1 expression is associated with improved outcome in human CML. These data demonstrate the BCR-ABL1-specific, cell-intrinsic pathways leading to altered interactions with the vascular niche via the modulation of adhesion molecules - which could be exploited therapeutically in the future.

B cell receptor-mediated antigen gathering requires ubiquitin ligase Cbl and adaptors Grb2 and Dok-3 to recruit dynein to the signaling microcluster.

The B cell receptor (BCR) mediates B cell antigen gathering and acquisition for presentation to T cells. Although the amount of antigen presentation to T cells determines the extent of B cell activation, the molecular mechanisms underlying antigen gathering remain unexplored. Here, through a combination of high-resolution imaging, genetics and quantitative mass spectrometry, we demonstrate that adaptors Grb2 and Dok-3, and ubiquitin ligase Cbl in signaling BCR microclusters mediate association with the microtubule motor dynein. Furthermore, we visualize the localization and movement of these microclusters on the underlying microtubule network. Importantly, disruption of this network or diminished dynein recruitment in Grb2-, Dok-3-, or Cbl-deficient B cells, does not influence microcluster formation or actin-dependent spreading, but abrogates directed movement of microclusters and antigen accumulation. Thus we identify a surprising but pivotal role for dynein and the microtubule network alongside Grb2, Dok-3, and Cbl in antigen gathering during B cell activation.

Leukemic blasts program bone marrow adipocytes to generate a protumoral microenvironment.

Despite currently available therapies, most patients diagnosed with acute myeloid leukemia (AML) die of their disease. Tumor-host interactions are critical for the survival and proliferation of cancer cells; accordingly, we hypothesize that specific targeting of the tumor microenvironment may constitute an alternative or additional strategy to conventional tumor-directed chemotherapy. Because adipocytes have been shown to promote breast and prostate cancer proliferation, and because the bone marrow adipose tissue accounts for up to 70% of bone marrow volume in adult humans, we examined the adipocyte-leukemia cell interactions to determine if they are essential for the growth and survival of AML. Using in vivo and in vitro models of AML, we show that bone marrow adipocytes from the tumor microenvironment support the survival and proliferation of malignant cells from patients with AML. We show that AML blasts alter metabolic processes in adipocytes to induce phosphorylation of hormone-sensitive lipase and consequently activate lipolysis, which then enables the transfer of fatty acids from adipocytes to AML blasts. In addition, we report that fatty acid binding protein-4 (FABP4) messenger RNA is upregulated in adipocytes and AML when in coculture. FABP4 inhibition using FABP4 short hairpin RNA knockdown or a small molecule inhibitor prevents AML proliferation on adipocytes. Moreover, knockdown of FABP4 increases survival in Hoxa9/Meis1-driven AML model. Finally, knockdown of carnitine palmitoyltransferase IA in an AML patient-derived xenograft model improves survival. Here, we report the first description of AML programming bone marrow adipocytes to generate a protumoral microenvironment.