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Genetic alterations in the DNA damage response (DDR) pathway are a frequent mechanism of resistance to chemoimmunotherapy (CIT) in B-cell malignancies. We have previously shown that the synergy of CIT relies on secretory crosstalk elicited by chemotherapy between the tumor cells and macrophages. Here, we show that loss of multiple different members of the DDR pathway inhibits macrophage phagocytic capacity in vitro and in vivo. Particularly, loss of TP53 led to decreased phagocytic capacity ex vivo across multiple B-cell malignancies. We demonstrate via in vivo cyclophosphamide treatment using the Eμ-TCL1 mouse model that loss of macrophage phagocytic capacity in Tp53-deleted leukemia is driven by a significant downregulation of a phagocytic transcriptomic signature using small conditional RNA sequencing. By analyzing the tumor B-cell proteome, we identified a TP53-specific upregulation of proteins associated with extracellular vesicles (EVs). We abrogated EV biogenesis in tumor B-cells via clustered regularly interspaced short palindromic repeats (CRISPR)-knockout (KO) of RAB27A and confirmed that the EVs from TP53-deleted lymphoma cells were responsible for the reduced phagocytic capacity and the in vivo CIT resistance. Furthermore, we observed that TP53 loss led to an upregulation of both PD-L1 cell surface expression and secretion of EVs by lymphoma cells. Disruption of EV bound PD-L1 by anti–PD-L1 antibodies or PD-L1 CRISPR-KO improved macrophage phagocytic capacity and in vivo therapy response. Thus, we demonstrate enhanced EV release and increased PD-L1 expression in TP53-deficient B-cell lymphomas as novel mechanisms of macrophage function alteration in CIT resistance. This study indicates the use of checkpoint inhibition in the combination treatment of B-cell malignancies with TP53 loss.

International audience; There is ample evidence in the epidemiological literature that polyphenols, the major non-vitamin antioxidants in plant foods and beverages, have a beneficial effect on heart disease. Until recently other mechanisms which polyphenols exhibit such as cell signaling and regulating nitric oxide bioavailability have been investigated. The oxidation theory of atherosclerosis implicates LDL oxidation as the beginning step in this process. Nine polyphenols from eight different classes and several of their O-methylether, O-glucuronide and O-sulfate metabolites have been shown in this study to bind to the lipoproteins and protect them from oxidation at lysosomal/inflammatory pH (5.2), and physiological pH (7.4). Polyphenols bind to the apoprotein at pH 7.4 with Kb > 106M- 1 and the number of molecules of polyphenols bound per LDL particle under saturation conditions varied from 0.4 for ferulic acid to 13.1 for quercetin. Competition studies between serum albumin and LDL show that substantial lipoprotein binding occurs even in the presence of a great molar excess of albumin, the major blood protein. These in vitro results are borne out by published human supplementation studies showing that polyphenol metabolites from red wine, olive oil and coffee are found in LDL even after an overnight fast. A single human supplementation with various fruit juices, coffee and tea also produced an ex vivo protection against lipoprotein oxidation under postprandial conditions. This in vivo binding is heart-protective based on published olive oil consumption studies. Relevant to heart disease, we hypothesize that the binding of polyphenols and metabolites to LDL functions as a transport mechanism to carry these antioxidants to the arterial intima, and into endothelial cells and macrophages. Extracellular and intracellular polyphenols and their metabolites are heartprotective by many mechanisms and can also function as potent “intraparticle” and intracellular antioxidants due to their localized concentrations that can reach as high as the micromolar level. Low plasma concentrations makepolyphenols and their metabolites poor plasma antioxidants but their concentration in particles such as lipoproteins and cells is high enough for polyphenols to provide cardiovascular protection by direct antioxidant effects and by other mechanisms such as cell signaling.

SummaryChemoimmunotherapy (CIT) is the standard of care in B-cell malignancies. It is relying on synergistic effects of alkylating chemotherapy and monoclonal antibodies via secretory crosstalk with effector macrophages. Here, we observed that loss of p53 function mediates resistance to CIT by suppressing macrophage phagocytic function.Loss of p53 leads to an upregulation of PDL1 and an increased formation of extracellular vesicles (EVs). EVs directly inhibit macrophage phagocytosis by PDL1 surface expression. Suppression of phagocytic function by lymphoma cell-derived EVs could be abrogated by pre-incubation of EVs with anti-PDL1 antibodies, CRISPR-KO ofPDL1and abrogation of EV formation byRAB27A-KO in lymphoma cells. Immune checkpoint inhibition represents a viable strategy to overcome EV-mediated resistance to chemoimmunotherapy in lymphoma.SignificanceLoss ofTP53mediates cell autonomous resistance to genotoxic chemotherapy, moreover non-cell autonomous effects may cause therapy resistance mediated by the tumor microenvironment. We identify aTP53-dependent mechanism that mediates resistance to synergistic chemoimmunotherapy by increasing formation of EVs and expression of the PDL1 immune checkpoint. PDL1 on EVs is directly responsible for macrophage suppression, preventing the exertion of the essential effector function of antibody-dependent cellular phagocytosis. This novel mechanism of resistance is in turn targetable by PDL1 checkpoint inhibition. Enhanced EV-release and immune checkpoint expression in lymphoma are novel mechanisms of macrophage modulation in the lymphoma microenvironment. We provide a novel principle of resistance to chemoimmunotherapy (CIT) representing of immediate relevance to treatment of refractory B-cell lymphoma.HighlightsLoss ofTP53in B-cell lymphoma induces resistance towards chemoimmunotherapy (CIT) by inhibition of macrophage effector function through PDL1 upregulationLoss ofTP53increases formation of extracellular vesicles (EVs) carrying PDL1EVs inhibit antibody-mediated cellular phagocytosis (ADCP), a key macrophage effector function in CITTargeting PDL1 on EVs with immune checkpoint inhibitors overcomesTP53-mediated resistance to CIT

Aldehyde dehydrogenases (ALDHs) are multifunctional enzymes that oxidize diverse endogenous and exogenous aldehydes. We conducted a meta-analysis based on The Cancer Genome Atlas and Gene Expression Omnibus data and detected genetic alterations in ALDH1A1, ALDH1A3, or ALDH3A1, 86% of which were gene amplification or mRNA upregulation, in 31% of nonsmall cell lung cancers (NSCLCs). The expression of these isoenzymes impacted chemoresistance and shortened survival times in patients. We hypothesized that these enzymes provide an oxidative advantage for the persistence of NSCLC. To test this hypothesis, we used genetic and pharmacological approaches with DIMATE, an irreversible inhibitor of ALDH1/3. DIMATE showed cytotoxicity in 73% of NSCLC cell lines tested and demonstrated antitumor activity in orthotopic xenografts via hydroxynonenal-protein adduct accumulation, GSTO1-mediated depletion of glutathione and increased H2O2. Consistent with this result, ALDH1/3 disruption synergized with ROS-inducing agents or glutathione synthesis inhibitors to trigger cell death. In lung cancer xenografts with high to moderate cisplatin resistance, combination treatment with DIMATE promoted strong synergistic responses with tumor regression. These results indicate that NSCLCs with increased expression of ALDH1A1, ALDH1A3, or ALDH3A1 may be targeted by strategies involving inhibitors of these isoenzymes as monotherapy or in combination with chemotherapy to overcome patient-specific drug resistance.

Cell fate determination during development often requires morphogen transport from producing to distant responding cells. Hedgehog (Hh) morphogens present a challenge to this concept, as all Hhs are synthesized as terminally lipidated molecules that form insoluble clusters at the surface of producing cells. While several proposed Hh transport modes tie directly into these unusual properties, the crucial step of Hh relay from producing cells to receptors on remote responding cells remains unresolved. Using wing development in Drosophila melanogaster as a model, we show that Hh relay and direct patterning of the 3–4 intervein region strictly depend on proteolytic removal of lipidated N-terminal membrane anchors. Site-directed modification of the N-terminal Hh processing site selectively eliminated the entire 3–4 intervein region, and additional targeted removal of N-palmitate restored its formation. Hence, palmitoylated membrane anchors restrict morphogen spread until site-specific processing switches membrane-bound Hh into bioactive forms with specific patterning functions., eLife digest Each cell in a developing embryo receives information that determines what type of body structure it will form. In fruit flies, this information is partly given by a protein called Hedgehog. In the embryo cells that receive it, Hedgehog can trigger a series of events which activate certain genes and thereby regulate structure formation. The Hedgehog proteins are produced by a different organizing group of cells: from there they transport within the embryo, creating a gradient. Depending on where a responding cell is in the embryo, it receives a different amount of Hedgehog, which gives the cell its identity. For example, Hedgehog proteins form a gradient across a fruit fly’s developing wing, which creates a visible vein pattern. How Hedgehog proteins form gradients is enigmatic, however, because once produced, they cling to the cells that created them. The reason for this unusual behavior is that the two ends of the Hedgehog protein are attached to a different fat molecule. In particular, one extremity is linked to a fat molecule called palmitate. These ends’ fatty additions anchor Hedgehog to the cells that produced them. Then, the tethered proteins gather together to form chain-like clusters where they inactivate each other: the extremity with the palmitate ‘hides’ the portion of the neighboring protein that binds to the receiving cells. It is still unclear how Hedgehog can be activated and released to reach these faraway cells. One hypothesis is that an enzyme comes to the clusters and frees the proteins by cutting both of Hedgehog’s fatty anchors. Thanks to how the palmitate tethers Hedgehog to the cell, the protein is positioned in such a way that when the enzyme makes its snip, the binding site on the neighboring Hedgehog gets exposed: this protein is activated and, when also cut by the enzyme, released. Here, Schürmann et al. create an array of mutant Hedgehog proteins – for example some without palmitate, some with palmitate that cannot be removed by the enzyme – and study how they affect the development of the wing’s pattern in the fruit fly. Coupled with the imaging of the clusters, these experiments support the hypothesis that the palmitate anchor is necessary so that Hedgehog proteins can be turned on before diffusing away. The Hedgehog family of proteins is also present in humans, where it presides over the development of the embryo but is also involved in cancer. Understanding how Hedgehog works in the fruit fly could lead to new discoveries in humans too.

Proteolytic processing of cell-surface-bound ligands, called shedding, is a fundamental system to control cell-cell signaling. Yet, our understanding of how shedding is regulated is still incomplete. One way to increase the processing of dual-lipidated membrane-associated Sonic hedgehog (Shh) is to increase the density of substrate and sheddase. This releases and also activates Shh by the removal of lipidated inhibitory N-terminal peptides from Shh receptor binding sites. Shh release and activation is enhanced by Scube2 [signal sequence, cubulin (CUB) domain, epidermal growth factor (EGF)-like protein 2], raising the question of how this is achieved. Here, we show that Scube2 EGF domains are responsible for specific proteolysis of the inhibitory Shh N-terminus, and that CUB domains complete the process by reversing steric masking of this peptide. Steric masking, in turn, depends on Ca

Proteolytic processing of cell-surface bound ligands, called shedding, is a fundamental system to control cell-cell signaling. Yet, our understanding of how shedding is regulated is still incomplete. One way to increase the processing of dual-lipidated membrane-associated Sonic hedgehog (Shh) is to increase the density of substrate and sheddase. This releases and also activates Shh by the removal of lipidated inhibitory N-terminal peptides from their receptor binding sites. Shh release and activation is enhanced by Scube2 [signal sequence, cubulin (CUB) domain, epidermal growth factor (EGF)-like protein 2], raising the question of how this is achieved. Here we show that Scube2 EGF domains prepare specific proteolysis of the inhibitory Shh N-terminus and that CUB domains complete the process by reversing steric masking of this peptide. Steric masking in turn depends on Ca++ occupancy of Shh ectodomains, unveiling a new mode of shedding regulation at the substrate level. Importantly, Scube2 uncouples Shh peptide processing from their lipid-mediated juxtamembrane positioning and thereby explains the long-standing conundrum that N-terminally unlipidated Shh shows patterning activity in Scube2-expressing vertebrates, but not in invertebrates that lack Scube orthologs.

Growth of chronic lymphocytic leukemia cells strongly depends on a nurturing microenvironmental niche that is specifically primed by diverse, bi-directional interactions to promote leukemic homing, proliferation and progression. The Src-family kinase Lyn was previously identified by our group as a key factor for the formation of this pro-leukemic niche and for the expansion of CLL cells, using the Eµ-TCL1 mouse model. In order to attribute the pro-leukemic function of Lyn to a specific cell type, chimeric mice with lineage-specific defects of Lyn within hematopoietic or non-hematopoietic compartments were generated by irradiating BL6J-mice lethally and restoring their hematopoietic system with Lyn-WT or Lyn-KO stem cells. Consecutively mice were xenotransplanted with TCL1+-malignant cells. Lyn deficiency within the non-hematopoietic compartment decelerated leukemic expansion to a higher degree than did Lyn deficiency within the hematopoietic compartment. Completely Lyn deficient mice showed a more prominent retardation of leukemic expansion compared with both lineage specific Lyn deficient mouse strains, suggesting an additive effect of the two distinct compartments for leukemic expansion. In focusing on the non-hematopoietic fibroblastic bystander cells, primary human CLL cells were cocultured in vitro with Lyn-deficient mouse embryonic fibroblasts as well as Lyn-KO human HS5 cells, generated via the CRISPR-Cas9 system, and leukemic cell survival was assessed over time. All Lyn-deficient fibroblasts showed a significantly reduced feeding capacity for CLL cells compared to WT stroma, indicating the functional relevance of Lyn in leukemia-associated fibroblasts. Subsequently, transcriptomic, proteomic and phosphoproteomic alterations related to Lyn-KO in HS5 cells were comprehensively analyzed, revealing a surprisingly extensive change in gene and protein expression pattern that appeared to be regulated mainly at the transcriptional level. The differentially expressed genes were remarkably often extracellular matrix (ECM)-, cytoskeleton- or cytokine-associated. GO-term enrichment analysis additionally suggested a correlation with ECM processes. Therefore, we hypothesized that Lyn-deficiency might induce transcriptional changes of the cancer-associated fibroblast (CAF)-like phenotype, thus leading to a reduction in leukemic feeding capacity. The diminished expression of several CAF-makers congruent with this reduced activation status was validated in Lyn-KO fibroblasts, as well as the transcriptionally regulated differential expression of chosen target genes. Amongst those, the deubiquitinating enzyme UCHL1 was most abundantly reduced in Lyn-KO HS5 cells, showing an almost complete loss of mRNA and protein expression. Application of a specific UCHL1-inhibitor -in a dose without toxic effects on CLL cells - to CLL-stroma coculture resulted in a significantly hampered feeder effect and reduced CLL cell survival, implying a functional relevance of microenvironmental UCHL1 for stromal support in our system. Additionally, stroma cell death induced by higher drug concentrations in WT cells was completely prohibited in Lyn-KO stroma, illustrating the importance of Lyn for regulating UCHL1 expression and function. In summary, we propose that the Lyn kinase contributes to the formation of a supportive microenvironment via the transcriptional reprogramming of stroma fibroblasts into a "CAF-like" phenotype, which echances viability of CLL cells. In addition, UCHL1 might be a potentially druggable mediator of this activation process. Disclosures Hallek: Roche, Gilead Sciences, Inc., Mundipharma, Janssen, Celgene, Pharmacyclics, AbbVie: Honoraria, Research Funding, Speakers Bureau.

Decision making in cellular ensembles requires the dynamic release of signaling molecules from the producing cells into the extracellular compartment. One important example of molecules that require regulated release in order to signal over several cell diameters is the Hedgehog (Hh) family, because all Hhs are synthesized as dual-lipidated proteins that firmly tether to the outer membrane leaflet of the cell that produces them. Factors for the release of the vertebrate Hh family member Sonic Hedgehog (Shh) include cell-surface sheddases that remove the lipidated terminal peptides, as well as the soluble glycoprotein Scube2 that cell-nonautonomously enhances this process. This raises the question of how soluble Scube2 is recruited to cell-bound Shh substrates to regulate their turnover. We hypothesized that heparan sulfate (HS) proteoglycans (HSPGs) on the producing cell surface may play this role. In this work, we confirm that HSPGs enrich Scube2 at the surface of Shh-producing cells and that Scube2-regulated proteolytic Shh processing and release depends on specific HS. This finding indicates that HSPGs act as cell-surface assembly and storage platforms for Shh substrates and for protein factors required for their release, making HSPGs critical decision makers for Scube2-dependent Shh signaling from the surface of producing cells.

Pattern recognition represents an attractive scientific field in the study of biological interactions. The interactions between flavonoids and proteins are extremely important in the explanation of the biological activity of these compounds. In this paper, the geometrical morphology of pseudo-phase portrait of the primary sequence of several proteins is evaluated, in order to have an approach to the pattern recognition in flavonoid-protein interactions. The theoretical affinity order (TAO) among flavonoids and amino acids residues of proteins is additionally applied in order to reduce the protein sequence complexity by residue grouping. The high potentiality of pseudo-phase portrait, as a qualitative descriptor is shown. Its morphology suffers significant modifications as a function of the analyzed protein sequences and therefore it becomes an important indicator or a characteristic pattern of flavonoid-protein interactions. It could be a useful application in the prediction of potential proteins to interact with flavonoid molecules.

Sonic Hedgehog (Shh) is a representative of the evolutionary closely related class of Hedgehog proteins that have essential signaling functions in animal development. The N-terminal domain (ShhN) is also assigned to the group of LAS proteins (LAS = Lysostaphin type enzymes, D-Ala-D-Ala metalloproteases, Sonic Hedgehog), of which all members harbor a structurally well-defined center; however, it is remarkable that ShhN so far is the only LAS member without proven peptidase activity. Another unique feature of ShhN in the LAS group is a double- center close to the zinc. We have studied the effect of these calcium ions on ShhN structure, dynamics, and interactions. We find that the presence of calcium has a marked impact on ShhN properties, with the two calcium ions having different effects. The more strongly bound calcium ion significantly stabilizes the overall structure. Surprisingly, the binding of the second calcium ion switches the putative catalytic center from a state similar to LAS enzymes to a state that probably is catalytically inactive. We describe in detail the mechanics of the switch, including the effect on substrate co-ordinating residues and on the putative catalytic water molecule. The properties of the putative substrate binding site suggest that ShhN could degrade other ShhN molecules, e.g. by cleavage at highly conserved glycines in ShhN. To test experimentally the stability of ShhN against autodegradation, we compare two ShhN mutants in vitro: (1) a ShhN mutant unable to bind calcium but with putative catalytic center intact, and thus, according to our hypothesis, a constitutively active peptidase, and (2) a mutant carrying additionally mutation E177A, i.e., with the putative catalytically active residue knocked out. The in vitro results are consistent with ShhN being a cannibalistic zinc-peptidase. These experiments also reveal that the peptidase activity depends on ., Author Summary Hedgehog proteins are important “morphogens” that steer embryonic development in concentration-dependent ways. Despite many years of intensive research, the mechanism of morphogen action is still under debate. We have studied properties of ShhN, the actual signaling part of Sonic Hedgehog, by a comprehensive set of computational methods and based on experimentally determined molecular structures. Our work suggests surprising new features of ShhN, namely that ShhN is an enzyme that acts as a cannibalistic peptidase, and that this enzymatic function is switched off by the binding of calcium ions to ShhN. Our computational approach reveals the details of this novel switching mechanism. To test the predicted autodegradation of ShhN, we study in vitro the stability of specific mutants, and we find that these experiments are in agreement with predictions.