Myeloproliferative neoplasms (MPN) are a group of clonal disorders that affect hematopoietic stem/progenitor cells. These disorders are often caused by oncogenic driver mutations associated with ...persistent Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling. While JAK inhibitors, such as ruxolitinib, reduce MPN-related symptoms in myelofibrosis, they do not influence the underlying cause of the disease and are not curative. Due to these limitations, there is a need for alternative therapeutic strategies and targets. Heat shock proteins (HSPs) are cytoprotective stress-response chaperones involved in protein homeostasis and in many critical pathways, including inflammation. Over the last decade, several research teams have unraveled the mechanistic connection between STAT signaling and several HSPs, showing that HSPs are potential therapeutic targets for MPN. These HSPs include HSP70, HSP90 (chaperoning JAK2) and both HSP110 and HSP27, which are key factors modulating STAT3 phosphorylation status. Like the HSPs, the PD-1/PD-L1 signaling pathway has been widely studied in cancer, but the importance of PD-L1-mediated immune escape in MPN was only recently reported. In this review, we summarize the role of HSPs and PD-1/PD-L1 signaling, the modalities of their experimental blockade, and the effect in MPN. Finally, we discuss the potential of these emerging targeted approaches in MPN therapy.
Abstract
Multiple myeloma (MM) cells express TLR. It has been shown that TLR ligands induce the proliferation, survival, and immune surveillance escape of MM cells through MyD88-TLR pathways. ...Deciphering TLR function in MM cells will help in understanding the mechanisms of tumor cell growth. In this study, we examined the response of MM cells to the MyD88-independent/TIR-domain-containing adapter-inducing IFN-β-dependent TLR3. Deregulation of NF-κB pathway is a feature of MM cells, and we wondered whether TLR3 activation could mobilize the NF-κB pathway. We show that five of seven human myeloma cell line (HMCL) cells expressed TLR3. In the presence of the synthetic TLR3 ligand (poly(I:C)), activation of NF-κB pathway was observed in three of five selected TLR3+ HMCL, NCI-H929, RPMI 8226, and KMM1. In agreement with NF-κB activation, only these three HMCL responded to poly(I:C), although by either an increase (KMM1) or a decrease (NCI-H929, RPMI 8226) of proliferation. We show that KMM1 increase of proliferation was prevented by NF-κB inhibitor. In contrast, inhibition of proliferation in both NCI-H929 and RPMI 8226 was due to IFN-α-induced apoptosis. We next demonstrated that p38 MAPK pathway controlled both IFN-α secretion and IFN-α-mediated cell death. Moreover, cell death also involved activation of ERK1/2 pathway. In conclusion, our results show that TLR3 ligand induces NF-κB pathway activation in MM and support a switching function of type I IFN in the functional outcome of TLR3 triggering in tumor cells.
PIK3CA-related overgrowth spectrum is caused by mosaicism mutations in the PIK3CA gene. These mutations, which are also observed in various types of cancer, lead to a constitutive activation of the ...PI3K/AKT/mTOR pathway, increasing cell proliferation. Heat shock transcription factor 1 (HSF1) is the major stress-responsive transcription factor. Recent findings indicate that AKT phosphorylates and activates HSF1 independently of heat-shock in breast cancer cells. Here, we aimed to investigate the role of HSF1 in PIK3CA-related overgrowth spectrum. We observed a higher rate of proliferation and increased phosphorylation of AKT and p70S6K in mutant fibroblasts than in control cells. We also found elevated phosphorylation and activation of HSF1, which is directly correlated to AKT activation. Specific AKT inhibitors inhibit HSF1 phosphorylation as well as HSF1-dependent gene transcription. Finally, we demonstrated that targeting HSF1 with specific inhibitors reduced the proliferation of mutant cells. As there is currently no curative treatment for PIK3CA-related overgrowth spectrum, our results identify HSF1 as a new potential therapeutic target.
•PIK3CA-related overgrowth spectrum is characterized by increased cell proliferation.•Strong activation of HSF1 is found in PIK3CA-related overgrowth spectrum fibroblasts.•AKT inhibitors reduces HSF1 activation and HSF1-dependent gene transcription.•HSF1 inhibition blocks the PIK3CA-related overgrowth spectrum proliferation.
Cohen syndrome (CS) is a rare autosomal recessive disorder with multisytemic clinical features due to mutations in the VPS13B gene, which has recently been described encoding a mandatory membrane ...protein involved in Golgi integrity. As the Golgi complex is the place where glycosylation of newly synthesized proteins occurs, we hypothesized that VPS13B deficiency, responsible of Golgi apparatus disturbance, could lead to glycosylation defects and/or mysfunction of this organelle, and thus be a cause of the main clinical manifestations of CS. The glycosylation status of CS serum proteins showed a very unusual pattern of glycosylation characterized by a significant accumulation of agalactosylated fucosylated structures as well as asialylated fucosylated structures demonstrating a major defect of glycan maturation in CS. However, CS transferrin and α1-AT profiles, two liver-derived proteins, were normal. We also showed that intercellular cell adhesion molecule 1 and LAMP-2, two highly glycosylated cellular proteins, presented an altered migration profile on SDS-PAGE in peripheral blood mononuclear cells from CS patients. RNA interference against VPS13B confirmed these glycosylation defects. Experiments with Brefeldin A demonstrated that intracellular retrograde cell trafficking was normal in CS fibroblasts. Furthermore, early endosomes were almost absent in these cells and lysosomes were abnormally enlarged, suggesting a crucial role of VPS13B in endosomal-lysosomal trafficking. Our work provides evidence that CS is associated to a tissue-specific major defect of glycosylation and endosomal-lysosomal trafficking defect, suggesting that this could be a new key element to decipher the mechanisms of CS physiopathology.
Interleukin-6 (IL-6), although often regarded as a B-cell differentiation factor, was recently described as the essential survival factor for human plasmablasts in vivo in reactive plasmacytosis. The ...present study reinvestigated the roles of IL-6 and IL-2 in the generation of plasma cells from human memory B cells in vitro. The cells involved in this differentiation process were identified as preplasmablasts (CD20±CD38±CD138−), plasmablasts (CD20−CD38++CD138−), and early plasma cells (CD20−CD38+++CD138+++). IL-2 or IL-10 induced a strong generation of plasmablasts and early plasma cells (PCs). Compared to IL-2 or IL-10, IL-6 alone was inefficient at PC generation. However, when combined with IL-2 or IL-10, IL-6 enhanced generation of early PCs. Moreover, anti–IL-6 monoclonal antibody markedly reduced IL-2–induced generation of early plasma cells, but not of plasmablasts. These roles of IL-2 and IL-6 were consistent with the difference in the expression of their respective receptors (R). CD25 (IL-2Rα) was increased 72 ± 10-fold on activated B cells, but decreased and then disappeared on plasmablasts. Conversely, CD126 (IL-6Rα) was barely expressed on activated B cells, but increased 18 ± 2-fold on preplasmablasts. Finally, IL-6 enhanced the proliferation (2-fold increase) of IL-2–generated plasmablasts. In conclusion, the data indicate that IL-6 is a growth factor for nonmalignant human plasmablasts.
Hypomethylated CpG oligodeoxynucleotides (CpG ODNs) target TLR9 expressed by immune cells and are currently being evaluated as adjuvants in clinical trials. However, TLR signaling can promote some ...tumor growth and immune evasion, such as in multiple myeloma (MM). Therefore, deciphering the effects of CpG ODNs on cancer cells will help in preventing these adverse effects and in designing future clinical trials. TLR activation induces multiple signaling pathways, notably NF-kappaB that has been involved in the resistance to TRAIL. Thus, we wondered if CpG ODNs could modulate TRAIL-induced apoptosis in different models of tumors. Here, we show that TLR9+ (NCI-H929, NAN6, KMM1) and TLR9- MM cells (MM1S) were protected by CpG ODNs against recombinant TRAIL-induced apoptosis. By using two fully human, agonist mAbs directed against TRAIL receptors DR4 and DR5 (mapatumumab and lexatumumab, respectively), we show that the protection was restricted to DR5-induced apoptosis. Similar results were observed for two colon cancer (C45 and Colo205) and two breast cancer cell lines (HCC1569 and Cal51). The protection of CpG ODNs was mediated by its nuclease-resistant phosphorothioate backbone independent of TLR9. We next demonstrated by surface plasmon resonance that phosphorothioate-modified CpG ODNs directly bound to either TRAIL or lexatumumab and then decreased their binding to DR5. Finally, NK cell lysis of a DR5-sensitive MM cell line (NCI-H929) through TRAIL was partially inhibited by phosphorothioate-modified CpG ODNs. In conclusion, our results suggest that the phosphorothioate modification of CpG ODNs could dampen the clinical efficacy of CpG ODN-based adjuvants by altering TRAIL/TRAIL receptor interaction.
The XPO1 gene encodes exportin 1 (XPO1) that controls the nuclear export of cargo proteins and RNAs. Almost 25% of primary mediastinal B-cell lymphoma (PMBL) and classical Hodgkin lymphoma (cHL) ...cases harboured a recurrent XPO1 point mutation (NM_003400, chr2:g61718472C>T) resulting in the E571K substitution within the hydrophobic groove of the protein, the site of cargo binding. We investigated the impact of the XPO1E571K mutation using PMBL/cHL cells having various XPO1 statuses and CRISPR–Cas9-edited cells in which the E571K mutation was either introduced or knocked-out. We first confirmed that the mutation was present in both XPO1 mRNA and protein. We observed that the mutation did not modify the export capacity but rather the subcellular localisation of XPO1 itself. In particular, mutant XPO1 bound to importin β1 modified the nuclear export/import dynamics of relevant cargoes.
Cohen Syndrome (CS) is a rare autosomal recessive disorder, with defective glycosylation secondary to mutations in the VPS13B gene, which encodes a protein of the Golgi apparatus. Besides congenital ...neutropenia, retinopathy and intellectual deficiency, CS patients are faced with truncal obesity. Metabolism investigations showed abnormal glucose tolerance tests and low HDL values in some patients, and these could be risk factors for the development of diabetes mellitus and/or cardiovascular complications. To understand the mechanisms involved in CS fat storage, we used two models of adipogenesis differentiation: (i) SGBS pre-adipocytes with VPS13B invalidation thanks to siRNA delivery and (ii) CS primary fibroblasts. In both models, VPS13B invalidation led to accelerated differentiation into fat cells, which was confirmed by the earlier and increased expression of specific adipogenic genes, consequent to the increased response of cells to insulin stimulation. At the end of the differentiation protocol, these fat cells exhibited decreased AKT2 phosphorylation after insulin stimulation, which suggests insulin resistance. This study, in association with the in-depth analysis of the metabolic status of the patients, thus allowed us to recommend appropriate nutritional education to prevent the occurrence of diabetes mellitus and to put forward recommendations for the follow-up of CS patients, in particular with regard to the development of metabolic syndrome. We also suggest replacing the term obesity by abnormal fat distribution in CS, which should reduce the number of inappropriate diagnoses in patients who are referred only on the basis of intellectual deficiency associated with obesity.
Cohen syndrome (CS) is a rare genetic disorder due to mutations in
VPS13B
gene. Among various clinical and biological features, CS patients suffer from inconsistent neutropenia, which is associated ...with recurrent but minor infections. We demonstrate here that this neutropenia results from an exaggerate rate of neutrophil apoptosis. Besides this increased cell death, which occurs in the absence of any endoplasmic reticulum stress or defect in neutrophil elastase (ELANE) expression or localization, all neutrophil functions appeared to be normal. We showed a disorganization of the Golgi apparatus in CS neutrophils precursors, that correlates with an altered glycosylation of ICAM-1 in these cells, as evidenced by a migration shift of the protein. Furthermore, a striking decrease in the expression of
SERPINB1
gene, which encodes a critical component of neutrophil survival, was detected in CS neutrophils. These abnormalities may account for the excessive apoptosis of neutrophils leading to neutropenia in CS.
Key messages
Cohen syndrome patients’ neutrophils display normal morphology and functions.
Cohen syndrome patients’ neutrophils have an increased rate of spontaneous apoptosis compared to healthy donors’ neutrophils.
No ER stress or defective ELA2 expression or glycosylation was observed in Cohen syndrome patients’ neutrophils.
SerpinB1 expression is significantly decreased in Cohen syndrome neutrophils as well as in VPS13B-deficient cells.
Gap junctional communication between cancer cells and blood capillary cells is crucial to tumor growth and invasion. Gap junctions may transfer microRNAs (miRs) among cells. Here, we explore the ...impact of such a transfer in co-culture assays, using the antitumor miR-145 as an example. The SW480 colon carcinoma cells form functional gap junction composed of connexin-43 (Cx43) with human microvascular endothelial cells (HMEC). When HMEC are loaded with miR-145-5p mimics, the miR-145 level drastically increases in SW480. The functional inhibition of gap junctions, using either a gap channel blocker or siRNA targeting Cx43, prevents this increase. The transfer of miR-145 also occurs from SW480 to HMEC but not in non-contact co-cultures, excluding the involvement of soluble exosomes. The miR-145 transfer to SW480 up-regulates their Cx43 expression and inhibits their ability to promote angiogenesis. Our results indicate that the gap junctional communication can inhibit tumor growth by transferring miRs from one endothelial cell to neighboring tumor cells. This "bystander" effect could find application in cancer therapy.
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