Role of calcium signaling in chronic myeloid leukemia

La Leucémie Myéloïde Chronique (LMC) est une maladie clonale caractérisée par la présence du chromosome Philadelphie codant pour Bcr-Abl, une tyrosine kinase constitutivement active responsable de la leucémogenèse. Bien que très efficaces, les inhibiteurs de tyrosine kinase (ITKs) restent cependant inactifs sur les cellules souches leucémiques. Ce travail de thèse montre que la signalisation calcique, connue pour réguler de nombreux processus dans les cellules saines et cancéreuses, est importante dans la signalisation cellulaire au décours de la LMC. Le rôle des entrées calciques dépendantes des stocks (SOCEs) médiées par STIM1 (STromal Interaction Molecule 1) et les canaux Orai1 et TRPC1 ainsi que des entrées calciques induites par la thrombine a été étudié dans la leucémogenèse. Nous avons observé une diminution de ces entrées dans les cellules exprimant Bcr-Abl pouvant être expliquée par le changement de stœchiométrie Orai1/STIM1. Ceci entraîne la diminution de l'activation de NFAT (Nuclear Factor of Activated T-cells) ainsi que des conséquences sur la prolifération et la migration cellulaire mais pas sur l'apoptose. De plus, les SOCEs sont restaurées dans les cellules cancéreuses après traitement à l'Imatinib, le principal ITK. Nous proposons alors que l'expression de Bcr-Abl joue un rôle sur l'homéostasie calcique en entraînant une dérégulation générale des fonctions cellulaires dans les cellules leucémiques notamment via la voie PKC (Protein Kinase C). Ainsi, ces résultats montrent une dérégulation des entrées calciques dans les cellules exprimant Bcr-Abl, suggérant que la signalisation calcique puisse être une cible thérapeutique en parallèle avec les ITKs.Chronic Myeloid Leukemia (CML) is a clonal disease characterized by the presence of the Philadelphia chromosome encoding for Bcr-Abl, a constitutively active tyrosine kinase responsible for leukemogenesis. Although Bcr-Abl tyrosine kinase inhibitors (TKIs) have revolutionized the therapy of Ph+ leukemia, the complete eradication of CML is limited by the emergence of resistance in hematopoietic stem cells. This thesis proposes that calcium (Ca2+) signaling pathways, known to govern a large number of functions in normal and cancer cells, may be important in CML cell signaling. Therefore, we studied the role of Store Operated-Calcium entry (SOCE) (i.e. STromal Interaction Molecule 1 (STIM1), Orai1 and TRPC1 channels) and thrombin induced Ca2+ entry in leukemogenesis. We found a decrease in both calcium entries in Bcr-Abl-expressing cells compared to normal cells. The reduced SOCE seems related to a change in stoichiometry of Orai1/STIM1. This leads to a reduction of the Nuclear Factor of Activated T-cells (NFAT) translocation and functional consequences on cell proliferation and migration but not on apoptosis. Moreover, we showed that SOCE is restored in malignant cells after treatment with Imatinib, the main TKI. We proposed that Bcr-Abl expression could impact on Ca2+ homeostasis enhancing a general disorganization of cell functions in leukemia cells notably via Protein Kinase C (PKC) pathway. Altogether this work shows a deregulation of Ca2+ entry in Bcr-Abl-expressing cells, suggesting that the Ca2+ signaling pathway could be a therapeutic target in parallel with TKIs

Loss of Transient Receptor Potential Melastatin 3 ion channel function in natural killer cells from Chronic Fatigue Syndrome/Myalgic Encephalomyelitis patients

Abstract Background Chronic Fatigue Syndrome (CFS)/ Myalgic Encephalomyelitis (ME) is a debilitating disorder that is accompanied by reduced cytotoxic activity in natural killer (NK) cells. NK cells are an essential innate immune cell, responsible for recognising and inducing apoptosis of tumour and virus infected cells. Calcium is an essential component in mediating this cellular function. Transient Receptor Potential Melastatin 3 (TRPM3) cation channels have an important regulatory role in mediating calcium influx to help maintain cellular homeostasis. Several single nucleotide polymorphisms have been reported in TRPM3 genes from isolated peripheral blood mononuclear cells, NK and B cells in patients with CFS/ME and have been proposed to correlate with illness presentation. Moreover, a significant reduction in both TRPM3 surface expression and intracellular calcium mobilisation in NK cells has been found in CFS/ME patients compared with healthy controls. Despite the functional importance of TRPM3, little is known about the ion channel function in NK cells and the epiphenomenon of CFS/ME. The objective of the present study was to characterise the TRPM3 ion channel function in NK cells from CFS/ME patients in comparison with healthy controls using whole cell patch-clamp techniques. Methods NK cells were isolated from 12 age- and sex-matched healthy controls and CFS patients. Whole cell electrophysiology recording has been used to assess TRPM3 ion channel activity after modulation with pregnenolone sulfate and ononetin. Results We report a significant reduction in amplitude of TRPM3 current after pregnenolone sulfate stimulation in isolated NK cells from CFS/ME patients compared with healthy controls. In addition, we found pregnenolone sulfate-evoked ionic currents through TRPM3 channels were significantly modulated by ononetin in isolated NK cells from healthy controls compared with CFS/ME patients. Conclusions TRPM3 activity is impaired in CFS/ME patients suggesting changes in intracellular Ca2+ concentration, which may impact NK cellular functions. This investigation further helps to understand the intracellular-mediated roles in NK cells and confirm the potential role of TRPM3 ion channels in the aetiology and pathomechanism of CFS/ME

Rituximab impedes natural killer cell function in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis patients: A pilot in vitro investigation

Abstract Background A recent in vitro pilot investigation reported Rituximab significantly reduced natural killer (NK) cell cytotoxicity in healthy donors. Chronic fatigue syndrome/Myalgic encephalomyelitis (CFS/ME) is a debilitating disorder of unknown etiology. A consistent finding is a significant reduction in NK cell cytotoxicity. Rituximab has been reported having questionable potential therapeutic benefits for the treatment of CFS/ME, however, the potential effects of Rituximab on NK cell cytotoxicity in CFS/ME patients are yet to be determined. Methods A total of eight CFS/ME patients (48.63 ± 15.69 years) and nine non-fatigued controls (NFC) (37.56 ± 11.06 years) were included using the Fukuda case definition. Apoptotic function, lytic proteins and degranulation markers were measured on isolated NK cells using flow cytometry following overnight incubation with Rituximab at 10 μg/ml and 100 μg/ml. Results There was a significant reduction in NK cell lysis between CFS/ME patients and NFC following incubation with Rituximab at 100 μg/ml at 12.5:1 and 6.25:1 effecter-target (E:T) ratios (p < 0.05). However, there was no significant difference for NFC following incubation with Rituximab at 10 μg/ml and 100 μg/ml. There was no significant difference between CFS/ME patients and NFC for granzyme A and granzyme B prior to incubation with Rituximab and following overnight incubation with Rituximab at 10 μg/ml. There was a significant decrease in granzyme B in CFS/ME patients compared to NFC with 100 μg/ml of Rituximab prior to K562 cells stimulation (p < 0.05). There was a significant increase in CD107a (p < 0.05) and CD107b expression (p < 0.01) in NFC after stimulation with K562 cells prior to incubation with Rituximab. There was a significant increase in CD107b expression between CFS/ME patients and NFC prior to incubation with Rituximab and without stimulation of K562 cells (p < 0.01). Importantly, there was a significant increase in CD107b following overnight incubation with 100 μg/ml of Rituximab in NFC prior to K562 cells stimulation (p < 0.01). Conclusion This study reports significant decreases in NK cell lysis and a significant increase in NK cell degranulation following Rituximab incubation in vitro in CFS/ME patients, suggesting Rituximab may be toxic for NK cells. Caution should be observed in clinical trials until further investigations in a safe and controlled in vitro setting are completed

Deregulation of calcium homeostasis in Bcr-Abl-dependent chronic myeloid leukemia

International audienceBackground: Chronic myeloid leukemia (CML) results from hematopoietic stem cell transformation by the bcr-abl chimeric oncogene, encoding a 210 kDa protein with constitutive tyrosine kinase activity. In spite of the efficiency of tyrosine kinase inhibitors (TKI; Imatinib), other strategies are explored to eliminate CML leukemia stem cells, such as calcium pathways. Results: In this work, we showed that Store-Operated Calcium Entry (SOCE) and thrombin induced calcium influx were decreased in Bcr-Abl expressing 32d cells (32d-p210). The 32d-p210 cells showed modified Orai1/STIM1 ratio and reduced TRPC1 expression that could explain SOCE reduction. Decrease in SOCE and thrombin induced calcium entry was associated to reduced Nuclear Factor of Activated T cells (NFAT) nucleus translocation in 32d-p210 cells. We demonstrated that SOCE blockers enhanced cell mobility of 32d-p210 cells and reduced the proliferation rate in both 32d cell lines. TKI treatment slightly reduced the thrombin-induced response, but imatinib restored SOCE to the wild type level. Bcr-Abl is also known to deregulate Protein Kinase C (PKC), which was described to modulate calcium entries. We showed that PKC enhances SOCE and thrombin induced calcium entries in control cells while this effect is lost in Bcr-Abl-expressing cells. Conclusion: The tyrosine kinase activity seems to regulate calcium entries probably not directly but through a global cellular reorganization involving a PKC pathway. Altogether, calcium entries are deregulated in Bcr-Abl-expressing cells and could represent an interesting therapeutic target in combination with TKI

Systematic Review of Sleep Characteristics in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome

(1) Background—Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a multifaceted illness characterized by profound and persistent fatigue unrelieved by rest along with a range of other debilitating symptoms. Experiences of unrefreshing and disturbed sleep are frequently described by ME/CFS patients. This is the first systematic review assessing sleep characteristics in ME/CFS. The aim of this review is to determine whether there are clinical characteristics of sleep in ME/CFS patients compared to healthy controls using objective measures such as polysomnography and multiple sleep latency testing. (2) Methods—the following databases—Pubmed, Embase, Medline (EBSCO host) and Web of Science, were systematically searched for journal articles published between January 1994 to 19 February 2021. Articles that referred to polysomnography or multiple sleep latency testing and ME/CFS patients were selected, and further refined through use of specific inclusion and exclusion criteria. Quality and bias were measured using the Joanna Briggs Institute checklist. (3) Results—twenty observational studies were included in this review. The studies investigated objective measures of sleep quality in ME/CFS. Subjective measures including perceived sleep quality and other quality of life factors were also described. (4) Conclusions—Many of the parameters measured including slow- wave sleep, apnea- hypopnea index, spectral activity and multiple sleep latency testing were inconsistent across the studies. The available research on sleep quality in ME/CFS was also limited by recruitment decisions, confounding factors, small sample sizes and non-replicated findings. Future well-designed studies are required to understand sleep quality in ME/CFS patients

A preliminary investigation of nutritional intake and supplement use in Australians with myalgic encephalomyelitis/chronic fatigue syndrome and the implications on health-related quality of life

Background: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex, multisystem illness without a currently recognized pharmacological treatment. Dietary supplementation and modification have been posited as potential management strategies; however, their efficacy is controversial. Objective: This study aimed to assess the nutritional intake and supplement use of Australian ME/CFS patients and the perceived effect on health-related quality of life (HRQoL) for the first time in an Australian patient population. Design: Between February 2019 and January 2020, ME/CFS patients across Australia volunteered in this cross-sectional study in response to online advertisements. Eligible respondents were invited to complete three online self-administered questionnaires investigating their supplement use, nutritional intake, and HRQoL. The study participants’ supplement use and nutritional intake were summarized and compared with the population data returned from the Australian Health Survey (2011–2012). Multiple linear regression analysis was also performed to determine the effect of participants’ supplement use and nutrient intake on HRQoL. Results: Twenty-four eligible ME/CFS patients (54.2% meeting the International Consensus Criteria, 79.2% female, mean age = 43.4 ± 10.5 years) completed the online questionnaires. Supplement use was highly prevalent among the study sample (87.5%) and considerably more common when compared with population data (31.9%). Daily total fats and caffeine intakes were significantly higher among ME/CFS patients when compared with the Australian population (P = 0.009 and P = 0.033, respectively), whereas daily intakes of total carbohydrates and alcohol were significantly lower (both P < 0.001). No consistent trends between nutrition and supplement use with patients’ HRQoL could be identified. Conclusions: The daily diet and supplement use of ME/CFS patients appear to vary considerably from those of the general Australian population. Although the role of nutritional intake and supplement use on ME/CFS patients’ HRQoL remains unclear, dietary changes and the use of supplements appear to be of value to ME/CFS patients

A systematic review of neurological impairments in myalgic encephalomyelitis/ chronic fatigue syndrome using neuroimaging techniques.

BACKGROUND:Myalgic encephalomyelitis/ Chronic Fatigue Syndrome (ME/CFS) is a multi-system illness characterised by a diverse range of debilitating symptoms including autonomic and cognitive dysfunction. The pathomechanism remains elusive, however, neurological and cognitive aberrations are consistently described. This systematic review is the first to collect and appraise the literature related to the structural and functional neurological changes in ME/CFS patients as measured by neuroimaging techniques and to investigate how these changes may influence onset, symptom presentation and severity of the illness. METHODS:A systematic search of databases Pubmed, Embase, MEDLINE (via EBSCOhost) and Web of Science (via Clarivate Analytics) was performed for articles dating between December 1994 and August 2019. Included publications report on neurological differences in ME/CFS patients compared with healthy controls identified using neuroimaging techniques such as magnetic resonance imaging, positron emission tomography and electroencephalography. Article selection was further refined based on specific inclusion and exclusion criteria. A quality assessment of included publications was completed using the Joanna Briggs Institute checklist. RESULTS:A total of 55 studies were included in this review. All papers assessed neurological or cognitive differences in adult ME/CFS patients compared with healthy controls using neuroimaging techniques. The outcomes from the articles include changes in gray and white matter volumes, cerebral blood flow, brain structure, sleep, EEG activity, functional connectivity and cognitive function. Secondary measures including symptom severity were also reported in most studies. CONCLUSIONS:The results suggest widespread disruption of the autonomic nervous system network including morphological changes, white matter abnormalities and aberrations in functional connectivity. However, these findings are not consistent across studies and the origins of these anomalies remain unknown. Future studies are required confirm the potential neurological contribution to the pathology of ME/CFS

Additional file1: of Rituximab impedes natural killer cell function in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis patients: A pilot in vitro investigation

Figure S1. Representative flow cytometric plot of NK cell cytotoxic activity. PKH-26 was used to gate NK cells. PKH-26 negative cells were identified as K562 target cells and used to determine apoptosis. Annexin V was used to determine events undergoing apoptosis and 7-AAD was used to determine cells undergoing late apoptosis. Figure S2. Representative flow cytometric plot of intracellular staining analysis of lytic proteins. NK cells were isolated and were used is isolation purity was ≥95%. NK cells were selectively gated on using flow cytometry and isotype controls were used to determine the positive population for Granzyme A and Granzyme B expression. Figure S3. Representative flow cytometry plots for degranulation markers. NK cells were isolated and were used if isolation purity was ≥95%. NK cells were gated and selected using flow cytometry to determine CD107a and CD107b expression. Isotype controls were used to determine the positive population. (PDF 231 kb