Astonishingly subtle temporal and spatial regulations of Ca2+ transport proteins have been shown to elicit precise sequences of processes that have real physiological importance.,2+. Microgradients occur within spatial dimensions of 1–10 µm, and it became possible to visualize such events many years ago. Nanogradients, as the name implies, occur within much smaller spatial dimensions, typically decaying with space constants of tens of nanometers. Because the nanogradients may also be short-lasting, it is particularly challenging to visualize them.2+ signals are oscillatory and a locally elevated [Ca2+] returns quickly to the basal level due to diffusion away from the domain, reuptake by Ca2+ pumps into the endoplasmic reticulum (ER), uptake via the Ca2+ uniporter into the mitochondria, and extrusion to the extracellular space via Ca2+ pumps in the plasma membrane.,We have known for a long time that the physiologically important cytosolic Ca2+ signal generation near IP3 receptors (IP3Rs) on the ER membrane.,2+ release from the ER via IP3Rs is opening of Ca2+-release activated Ca2+ (CRAC) channels in the plasma membrane.2+] beneath the plasma membrane, particularly during activated Ca2+ influx.2+ domains in the subplasmalemmal space near CRAC channels and the processes initiated by such local signals have provided important insights into the control of many different physiological events.,In the electrically nonexcitable cells, the original focus was predominantly on local cytosolic Ca2+ signals are mostly oscillatory, but, in an important article published in this issue of Function,2+] in the local cytosolic space near the inner mouth of the CRAC channels, in spite of the bulk cytosolic Ca2+ concentration only being minimally elevated due to the operation of the plasma membrane Ca2+ ATPase.As mentioned above, it has generally been thought that physiological Caet al.2+ signaling system, thereby creating a situation that allows rigorous testing of the hypothesis that a sustained elevated subplasmalemmal [Ca2+] can be maintained with only minimal elevation of the bulk cytosolic [Ca2+], thus creating a standing Ca2+ gradient. In HEK293 cells, thapsigargin was used to arrest the ER Ca2+ pumps. Ca2+ therefore leaked out of the ER, gradually emptying the store and activated CRAC channels of the Orai1 type in the plasma membrane. In this situation, with the ER Ca2+ pumps out of action, the only transport proteins capable of removing excess Ca2+ from the cytosol would be the Ca2+ pumps in the plasma membrane. In addition to standard measurements of the bulk cytosolic [Ca2+], Barak et al.2+] in the subplasmalemmal space near the CRAC (Orai1) channels by expressing an Orai1 construct in which the Ca2+-sensitive fluorescent protein GECO was tagged to the N-terminus of the channel. The key experiments showed that thapsigargin elicited major rises in [Ca2+] both in the cytosol at large as well as close to the Orai1 channels but, whereas the bulk cytosolic [Ca2+] returned to a near prestimulation basal level within about 10–15 min, the local [Ca2+] close to the Orai1 channels remained markedly elevated even 25 min after the start of thapsigargin application. In spite of the relatively fast return of the bulk cytosolic [Ca2+] to the basal level, the elevated subplasmalemmal [Ca2+] was shown to be capable of driving the slowly developing migration of NFAT into the nucleus.2+ pumps, either by external La3+ or by increasing the external pH to 11, very effectively prevented the rapid return of the bulk cytosolic [Ca2+] to the basal level. In contrast, marked reduction of the external [Na+] had hardly any effect, indicating that the Na+–Ca2+ exchanger in the plasma membrane does not play any important role.2+ pumps in the ER, the plasma membrane Ca2+ pumps are by far the most important elements disposing of excess cytosolic Ca2+, in complete agreement with previous studies on electrically nonexcitable cells.,The experimental arrangement used by Barak 2+] in the subplasmalemmal space2+ pumps in the plasma membrane were colocalized with the CRAC channels. Indeed, Barak et al. show that during thapsigargin-elicited Ca2+ inflow, there is little colocalization between these two types of Ca2+ transporters in the HEK293 cells.2+ pumps are concentrated in the apical membrane, whereas the CRAC channels reside in the baso-lateral membrane, an arrangement of great physiological importance.The apparent prolonged existence of nanodomains with a high [Ca2+] in the subplasmalemmal domains and the bulk of the cytosol, it is necessary to assume a major “resistance” to Ca2+ movements. It has been known for a long time that Ca2+ diffusion in the cytosol is severely restricted.2+ diffuses more readily inside the ER than in the cytosol, as the Ca2+ binding capacity in the cytosol is orders of magnitude higher than in the lumen of the ER.In order to explain the sustained difference between  in both local domains and the bulk of the cytosol, but, as pointed out by Rizzuto and Pozzan,2+] in the cytosol depends exclusively on the equilibrium between Ca2+ entry and Ca2+ outflow across the plasma membrane. By exploiting a conceptually simple experimental situation, Barak et al.2+ pumps in the plasma membrane can protect the bulk of the cytosol against toxic elevations of [Ca2+] while still allowing a sustained local [Ca2+] elevation near the plasma membrane to drive gene expression. This is an achievement of real physiological importance.Are the conclusions from the experiments of Barak There are no data presented in this editorial/perspective.


TP53 mutation have higher immune infiltration and other immunomodulatory therapies could thus potentially be effective. Here, we performed the transcriptional analysis of distinct T‐cell subpopulations from TP53‐mutated AML to identify gene expression signatures suggestive of altered functional properties.Acute myeloid leukemia (AML) patients have limited effect from T‐cell‐based therapies, such as PD‐1 and CTLA‐4 blockade. However, recent data indicate that AML patients with + cytotoxic T lymphocytes (CTLs), conventional helper T cells (Th), and regulatory T cells (Tregs) were sorted from peripheral blood of AML patients with TP53 mutation (n = 5) and healthy donors (n = 3), using FACS, and the different subpopulations were subsequently subjected to RNA‐sequencing. Differentially expressed genes were identified and gene set enrichment analysis (GSEA) was performed to outline altered pathways and exhaustion status. Also, expression levels for a set of genes encoding established and emerging immuno‐oncological targets were defined.CD8TP53‐mutated AML as compared to control subjects. IFN‐α and IFN‐γ signaling were stronger in TP53‐mutated AML for both CTLs and Tregs. Furthermore, in TP53‐mutated AML as compared to healthy controls, Tregs showed gene expression signatures suggestive of metabolic adaptation to their environment, whereas CTLs exhibited features of exhaustion/dysfunction with a stronger expression of TIM3 as well as enrichment of a gene set related to exhaustion.The results showed altered transcriptional profiles for each of the T‐cell subpopulations from TP53‐mutated AML and open up for further exploration toward novel treatment regimens for these patients.The results provide insights on mechanisms underlying the inadequate immune response to leukemic cells in  Cytotoxic and regulatory T‐cells are transcriptionally altered in AML patients with TP53 mutation. The transcriptional profiles suggest metabolic adaptation and proliferation of Tregs, and exhaustion/dysfunction of cytotoxic T‐cells. Hence, these mechanisms could contribute to the inadequate immune response to leukemic cells and thus pave the way for novel treatment regimens for these patients. The results show transcriptional alterations suggestive of metabolic adaptation and proliferation for AML‐Tregs, whereas features of exhaustion were associated with AML‐CTLs. Furthermore, the gene encoding the exhaustion marker and emerging drug target TIM3 was expressed at higher levels by AML‐CTLs than in CTLs from healthy controls.In this study, we performed transcriptional profiling of different subpopulations of T‐cells  in a cohort of patients with 22.1TP53 mutation  at the local blood bank . The research was performed in accordance with relevant guidelines/regulations and was approved by the Regional Ethics Committee  and informed consent was obtained from all AML patients.Peripheral blood mononuclear cells (PBMCs) from untreated AML patients with 2.2+lineage−  cells. Out of T‐cells, CTLs (CD3+CD8+) and CD4+ T‐cells were gated. Then, Tregs were further identified as CD4+CD25+CD127−/dim cells and Th cells as the remaining CD4+ T‐cells. RNA was extracted from sorted T‐cells using Arcturus PicoPure RNA Isolation Kit  according to the manufacturer's instruction.Thawed cells were stained with fixable viability stain 620 (BD Biosciences) according to the manufacturer's protocol. Subsequently, cells were washed in cold PBS  + 2% Fetal Bovine Serum (GIBCO), then blocked with mouse IgG (Jackson ImmunoResearch), and finally stained with an antibody panel Table  for 20 m2.3RNA concentration and integrity were analyzed with the Agilent 2100 bioanalyzer (Agilent Technologies), using the RNA 6000 pico kit (Agilent Technologies). Amplified cDNA was prepared from the RNA using the SMART‐seq v4 ultra‐low input RNA kit (Takara Biosciences), and cDNA sequencing libraries were prepared using the Nextera Library DNA preparation kit . The libraries were sequenced by a NextSeq 500 (Illumina), using paired‐end 151 bp reads. The reads were aligned to the human reference genome hg19 using STAR 2.5.0a.2.4Gene expression profiles were analyzed using Omics Explorer 3.2 (Qlucore). To identify differentially expressed genes among specific T‐cell subpopulations from both AML patients and control subjects, ANOVA was performed , and principal component analysis (PCA) was used to visualize the data. Additionally, significantly differentially expressed genes between T‐cell subpopulations in AML as compared to the corresponding control subpopulation were identified using a two‐tailed t‐test with FDR <0.05.2.5+ T‐cells in progressive HIV  was performed to determine if a gene set of interest was statistically enriched in one condition compared to another.33.1TP53‐mutated AML, differentially expressed genes were investigated. In total, 3698 genes were found to be differentially expressed among the six subpopulations i.e., CTLs, Tregs, and Th cells from AML and healthy control subjects. Transcriptional differences among T‐cell subpopulations from AML patients and control subjects were visualized in a PCA plot , whereas no significant difference was detected in the expression of other molecules, including PD‐1 and CTLA‐4 that represent targets of approved drugs used for specific cancer patient groups. Also, TNFRSF4/OX40 showed higher expression in AML‐Tregs as compared to AML‐CTLs (FDR < 0.05).To evaluate the potential of established and/or emerging immuno‐oncological drugs to affect T‐cells in 3.3GSEA was performed using the Hallmark gene sets to obtain a broad overview of pathways differentially regulated in specific T‐cell subpopulations in the AML cohort as compared to the respective population in healthy controls. CTLs, Tregs, and Th cells from AML patients were shown to be enriched for genes associated with fatty acid metabolism, as compared to their respective control subpopulations. Table . AML‐TreIn addition to metabolic pathways, AML‐Tregs were found to upregulate cell cycle pathways (E2F target and G2M checkpoint signatures) as compared to control‐Tregs as well as compared to AML‐CTLs. Moreover, AML‐CTLs and AML‐Tregs were both enriched for genes associated with IFN‐α and IFN‐γ responses, as compared to the corresponding population in healthy controls. Furthermore, AML‐CTLs displayed a negative enrichment score for gene sets associated with hedgehog signaling and NF‐κB mediated TNF‐signaling, as compared to the corresponding control subpopulation.3.4TP53‐mutated AML, we performed GSEA using three different exhaustion signatures, i.e., a signature of exhausted CD8+ T‐cells during chronic infection (CD8+ T‐cells in progressive HIV patients) and signatures of exhausted CD8+ T‐cells infiltrating liver and melanoma tumors, respectively.+ T‐cells during chronic infection; however, significant enrichment was not observed for signatures based on exhausted CD8+ T‐cells from solid tumors  and downregulation of stimulatory molecules  were identified as compared to CTLs from healthy controls.TP53‐mutated AML. One could thus speculate that the exhaustion profile of CTLs may be distinct in TP53‐mutated AML as compared to AML with other mutations.The presented data also show that AML‐CTLs display transcriptional alterations suggestive of exhaustion/dysfunction. For example, AML‐CTLs were significantly enriched for a gene set related to exhaustion during chronic infection in humans,TP53 mutation have different metabolic features in comparison to control‐Tregs and/or to AML‐CTLs. AML‐Tregs showed enrichment of gene sets associated with glycolysis, fatty acid metabolism, and oxidative phosphorylation as compared to controls, thus suggesting that AML‐Tregs upregulate their production of energy based on both glucose and fatty acids in the AML‐setting. Interestingly, both glycolysis and fatty acid metabolism were recently suggested to be important for the expansion of Tregs in cancer, based on studies in a murine colon carcinoma model.TP53‐mutated AML, possibly supporting the poor disease outcome.The broad pathway analysis, using GSEA and the Hallmark gene sets, suggested that Tregs from patients with TP53‐mutated AML and healthy controls while applying stringent FDR (<0.05). Furthermore, to assess whether the observed differences are specific to TP53‐mutated AML or a characteristic feature also of other molecular subtypes of AML, additional studies investigating larger cohorts of AMLs are required. In addition, functional assessment of the T cell compartment in TP53‐mutated AML is needed to firmly establish altered functionality.Relevant limitations to consider in the current study include the limited size of the cohort and the patient group comprising de novo, secondary and treatment‐related AML. Still, clear transcriptomic differences were observed between TP53‐mutated AML, as compared to healthy controls, providing clues on mechanisms involved in the compromised immune response in this subtype of AML. The results suggest that AML‐Tregs alter their metabolic pathways and gain a metabolic advantage over AML‐CTLs. At the same time, AML‐CTLs appear to be functionally impaired, featuring exhaustion properties including higher expression of TIM3. Further exploration of these findings could hence open up for new therapeutic strategies for the treatment of TP53‐mutated AML, a subtype associated with dismal prognosis using current treatment regimens.In summary, we demonstrate altered transcriptional profiles of CTLs, Tregs, and Th cells in Thoas Fioretos is a scientific advisor and board member of Qlucore AB .The rest of the authors declare no financial or commercial conflict of interest.Involved in cell sorting, analyzed the data, produced figures, and drafted the manuscript: Milad Abolhalaj. Involved in data analysis and manuscript revision: Viktor Sincic. Planned RNA extraction and sequencing and performed bioinformatical processing of the data: Henrik Lilljebjörn. Involved in antibody panel development and cell sorting: Carl Sandén. Supported data analysis, contributed with bioinformatical consultation: Alar Aab. Involved in antibody panel development and cell sorting: Karin Hägerbrand. Contributed to the study design: Peter Ellmark. Contributed to the study design: Carl A. K. Borrebaeck. Contributed to the study design and with patient samples: Thoas Fioretos. Contributed to the study design, involved in antibody panel development and cell sorting, supervised data analysis as well as manuscript drafting: Kristina Lundberg. All authors contributed to the interpretation of results and were involved in analysis consultation as well as in the reviewing and editing of the manuscript. All authors approved the final version of the manuscript.The research was performed in accordance with relevant guidelines/regulations and was approved by the Regional Ethics Committee  and informed consent was obtained from all AML patients.FigureS 1Click here for additional data file.TableS 1Click here for additional data file.TableS 2Click here for additional data file.TableS 3Click here for additional data file.TableS 4Click here for additional data file.


Despite improvement of current treatment strategies and novel targeted drugs, relapse and treatment resistance largely determine the outcome for acute myeloid leukemia (AML) patients. To identify the underlying molecular characteristics, numerous studies have been aimed to decipher the genomic- and transcriptomic landscape of AML. Nevertheless, further molecular changes allowing malignant cells to escape treatment remain to be elucidated. Mass spectrometry is a powerful tool enabling detailed insights into proteomic changes that could explain AML relapse and resistance. Here, we investigated AML samples from 47 adult and 22 pediatric patients at serial time-points during disease progression using mass spectrometry-based in-depth proteomics. We show that the proteomic profile at relapse is enriched for mitochondrial ribosomal proteins and subunits of the respiratory chain complex, indicative of reprogrammed energy metabolism from diagnosis to relapse. Further, higher levels of granzymes and lower levels of the anti-inflammatory protein CR1/CD35 suggest an inflammatory signature promoting disease progression. Finally, through a proteogenomic approach, we detected novel peptides, which present a promising repertoire in the search for biomarkers and tumor-specific druggable targets. Altogether, this study highlights the importance of proteomic studies in holistic approaches to improve treatment and survival of AML patients. Over the lastdecades, research efforts in acute myeloid leukemia (AML) have to a great extent uncovered the inter- and intratumor heterogeneity at AML onset, resulting in improved risk classification , 2 and nProteomics provides direct molecular phenotype characterization of AML and hence could enable better identification of potential druggable targets. High resolution isoelectric focusing liquid chromatography mass spectrometry (HiRIEF LC-MS) is an in-depth proteomic approach that allows the detection of smaller variations in protein levels and low abundant proteins through reduction in peptide complexity by fractionation , 7. AddiA few studies have performed global proteomics and phosphoproteomics on AML patient material e.g., –17). The. The17])n = 42; relapse [R], n = 71; primary resistant [PR], n = 6) from 47 adult and 22 pediatric R/PR AML cases in an unbiased, MS-based analysis. We report higher levels at relapse of proteins involved in regulation of mitochondrial translation and peptide biosynthesis, as well as relapse-associated pro-inflammatory proteomic signatures. In addition, several splicing-related proteins showed higher levels at relapse compared to diagnosis. We further incorporated genomic [To shed further light on the proteomic landscape of AML cells, we profiled 119 proteomes , two had a prior diagnosis of myelodysplastic syndromes (MDS), and two had t-MDS-AML. Event-free survival (EFS) was measured as the time from diagnosis until first relapse or initial treatment failure, with the latter indicated by time = 0. The median length of EFS for relapse cases was 10.0 months (range: 1.1–126.0) for adults and 9.9 months (range: 2.3–19.1) for children . Inclusion criteria were AML cases with available R/PR material of sufficient quality and yield, which were accessible through various biobanks in the Nordic countries. Acute promyelocytic leukemia cases were excluded. Informed consent was obtained from all patients or their legal guardians according to the Declaration of Helsinki. The study was approved by the Uppsala Ethical Review Board (Sweden) and the Regional Ethical Committee South-East (Norway). Sixty-two of the patients had range: 2.–19.1 forCD34-expressing bone marrow (BM) cells from five healthy donors  were used as normal controls (“BM-controls”) for the proteomic and proteogenomic analyses  data from the same AML cohort (reported in ref. ). Only tn = 22 diagnosis/relapse pairs; pediatric: n = 16 diagnosis/relapse pairs). Only peptides with more than one peptide spectrum match (PSM) were considered.Limma was used to identify differences in the levels of novel peptides between diagnosis and relapse samples . A paireP < 0.05 for two-sided tests unless otherwise stated.Throughout this study, significance was defined as FDR < 0.01 for peptide identification and n = 42) and relapse (n = 71), as well as PR samples (n = 6), including 38 patient-matched diagnosis-relapse pairs, and four diagnosis-PR pairs . To discover recurrent proteome changes between diagnosis and relapse, a subset of proteins , as also previously reported [For an overview of the proteomes, we applied unsupervised principal component analysis (PCA) on all AML- and BM-control samples part of the cohort  in adult and pediatric cases, respectively  among the highest ranked proteins linked to relapse in adult AML patients Fig. . ElevateCR1  at relapse compared to patient-matched diagnosis samples both in adults  and children . Here, Next, we applied a proteogenomic approach by taking advantage of the access to cohort-matched WGS/WES and RNA-seq data for investigation of potential novel and mutated peptides present in the proteomic data  remained, with an intersection of 92 identical peptides found in both cohorts (Supplementary Tables n = 5), long non-coding RNAs (n = 2), antisense transcripts (n = 1), or to several potential transcripts simultaneously. For the pediatric cohort, 68 peptides (43.6%) were annotated to 55 unique protein-coding genes, with the remaining peptides being annotated to 22 pseudogenes (14.1%), two intergenic regions, one antisense transcript, and the rest to several potential transcripts simultaneously , intronic regions  or other genic regions  genes, a total of 370 novel peptides  passing the outlier threshold  with significantly different abundance between relapse- and patient-matched pre-treatment samples , we examined the overlap between alterations identified at the DNA- or RNA level and our proteomic data. For this, a similar approach as for detection of novel peptides was utilized. Tumor-derived sequences were analyzed at the peptide level by searching HiRIEF LC-MS spectra against a customized database of mutated peptides. To generate the customized database, six-frame translation of SNVs detected from the WGS/WES data  was appn = 12; pediatric: n = 2) were detected by searching our MS-dataset (Supplementary Tables GDI2 (GDP Dissociation Inhibitor 2) found in AML008-D and AML008-R1 by HiRIEF LC-MS, whereas only identified in AML008-D by WES analysis of these samples. Manual inspection of the WES reads by using Integrative Genomics Viewer [Based on the customized database containing mutated peptides, 14 SAAAs . UniqueR2 . HoweveGenomic, transcriptomic, epigenomic and proteomic data independently contribute to a better understanding of the biological differences between pre-treatment AML cells and their counterparts at relapse. However, a final goal would be to fully integrate data from several omic-levels. To this end, we integrated RNA-seq data with HiRIEF LC-MS-derived proteomic data to investigate tumor specific peptides. Through this proteogenomic approach, we identified 370 novel peptides and protein products representing, for instance, novel splice variants and translated pseudogenes and other non-protein-coding genomic regions, with 58 of these validated through another, smaller, AML MS-dataset  Fig. 5 –15. AmonAlthough out of the scope of this study, further analyses are needed to validate leukemia specificity among the detected novel peptides. Here, we included five individual BM-controls. As a result, a subset of the reported novel peptides was identified at the transcriptomic and/or proteomic level also in the BM-controls, highlighting the importance of including normal controls into multi-omics study approaches.Collectively, our proteogenomic study suggests that distinct protein profiles can be linked to relapse, and may be associated with therapy resistance in AML. The identified protein profiles, together with AML-specific neoantigens, represent the foundation for the exploration of novel drugs and for maximizing the benefits of current treatments; altogether with the aim of improving outcome of R/PR AML patients.Supplemental InformationDataset 1


PML – RARA and abnormal eosinophilic bone marrow proliferation in AML with inv(16) or t; CBFB – MYH11. As well, at the end of the 2000s, a new distinctive morphologic sign associated with a particular phenotype, cytogenetic and molecular profile in acute leukemia, has been described and defined the so‐called cup‐like blasts [The importance of flow cytometry immunophenotyping, cytogenetic and molecular analysis in diagnosis and prognosis of acute leukemia has greatly increased in recent years and tends to gradually replace morphological examination. Nevertheless, the latter still keeps a central place and should not be overlooked. Several correlations between morphological signs and cytogenetic and molecular abnormalities have been identified in acute myeloid leukemia (AML), including the well‐known “faggot cells” in acute promyelocytic leukemia with  with inv6 or t(16NPM1) mutation, with or without co‐occurring fms‐related tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutation [NPM1 mutation is one of the most common recurrent genetic lesion in AML and is relatively specific for AML [FLT3‐ITD mutation is associated with higher white blood cell (WBC) count, higher percentages of bone marrow blasts, and worse prognosis [These blasts with prominent nuclear invagination are strongly associated with AML with normal karyotype and nucleophosmin 1 , and extreme leukocytosis (617 × 109/L). Blast cells represented 97% of WBC on the blood film. They were medium/high size with high nuclear‐cytoplasmic ratio, round nucleus, and basophilic nongranular cytoplasm. Approximately one‐third of these cells had prominent round and translucent invagination, which overlapped more than 25% of the nucleus and therefore met the definition of cup‐like blasts , thrombocytopenia 64 × 1009/L, andKMT2A rearrangement in B‐ALL [KMT2A‐AFF1. Next‐generation sequencing revealed KRAS mutation . No mutation was detected in NPM1 or FLT3. These results converged to the diagnosis of B‐lymphoblastic leukemia (B‐ALL) with cup‐like morphology.Flow cytometric immunophenotyping Figure  showed sin B‐ALL . CytogenGiven the results of the full blood count, the patient was immediately transferred to the intensive care unit (ICU). Upon arrival in ICU, she presented right hemiplegia with facial paralysis. Computed tomography (CT) scan revealed a large brain hematoma without medical or surgical possibility of treatment. Despite appropriated intensive cares, the patient's neurological condition continued to deteriorate and she died few hours later.NPM1 or concurrent NPM1 and FLT3‐ITD mutations is well recognized and reported in numerous studies [NPM1 and/or FLT3‐ITD mutations is restricted to 60% [NPM1 and FLT3‐ITD mutations. Furthermore, cup‐like blasts must not be considered as a specific feature of AML. Two others B‐ALL with cup‐like blasts occurring in adult patients are reported in the literature [NPM1 and FLT3 mutations were not found in either. B‐ALL with t; BCR – ABL1 was finally diagnosed in the first case [TP53 and DNMT3A mutations [IKZF1 deletion [IKZF1 deletion in pediatric B‐ALL [The strong correlation between AML with cup‐like blasts and  studies , 3, 4, 5terature , 8. One terature , but leurst case , while tutations , 8. Computations , 8. One LL with t;22; BCR ic B‐ALL NPM1 and/or FLT3 mutations, but these rare presentations of B‐ALL remind us that the presence of cup‐like blasts, even in high proportion, is not a morphologic argument for assigning blasts in the myeloid lineage.Blasts with cup‐like morphology are commonly described in AML with KMT2A‐AFF1‐rearranged are usually associated with high WBC count and dysmorphic blast population with a variable proportion of blasts meeting the morphologic criteria of monoblasts and others of lymphoblasts. Most cases are B‐ALL, but AML and mixed‐phenotype are also reported [KMT2A‐AFF1‐rearranged diagnosed in our laboratory, and one presented a cup‐like morphology according the threshold of 10% .According to the World Health Organisation (WHO) classification , acute lKMT2A‐AFF1 or other cytogenetic or molecular signature in adult B‐ALL. But this finding might be underestimated. A retrospective study with further investigations in adults B‐ALL, as it was done in pediatric B‐ALL [Cases of B‐ALL in adult patients with cup‐like blasts are exceptionally described. Given our results and the heterogeneity of the cases reported Table , it is tic B‐ALL , be need


While voltage is often viewed as the single critical factor gating these channels, research is starting to reveal a more intricate scenario whereby their function is markedly tuned. This emerging concept will be the focus of this three-part review, the first part articulating the mechanistic foundation of contractile development in vascular smooth muscle. Part two will extend this foundational knowledge, introducing readers to functional coupling and how neighboring L-type Ca2+ channels work cooperatively through signaling protein complexes, to facilitate their open probability. The final aspect of this review will discuss the impact of L-type Ca2+ channel trafficking, a process tied to cytoskeleton dynamics. Cumulatively, this brief manuscript provides new insight into how voltage, along with channel cooperativity and number, work in concert to tune Ca2+ responses and smooth muscle contraction.Vascular smooth muscle contraction is intimately tied to membrane potential and the rise in intracellular Ca Moreover, experimental data demonstrates that reducing Ca2+ sparklets activity notably slows Ca2+ refilling of the sarcoplasmic reticulum  and PKA (hyperglycemia and diabetes type II) is notable for enhancing arterial tone  cooperative gating among neighboring L-type Ca2+ channels; and 2) stimulus-induced protein trafficking. Experimental research now defines how each mechanism is regulated by protein kinases, anchoring proteins, cytoskeletal structures, and initiating stimuli. Translating this knowledge into relevant biological settings is the next Frontier and one destined to intrigue the next generation of vascular biologists. Are, for example, the number and size of L-type Ca2+ channel clusters truly tuned in temporally concert with the changing physiological environment? Likewise, how do L-type Ca2+ channel clusters change in pathobiological settings like sepsis, where the proinflammatory environment progressively leads to circulatory collapse? This deeper understanding of L-type Ca2+ channels and its linkage to vascular tone is expected to reveal new conceptual avenues for therapeutic development.This mini-review summarizes current thinking on how the functional tuning of L-type Ca


Many Ca2+-sensing ion channel proteins have evolved to receive and propagate Ca2+ signals. Among them are the Ca2+-activated potassium channels, a large family of potassium channels activated by rises in cytosolic calcium in response to Ca2+ influx via Ca2+-permeable channels that open during the action potential or Ca2+ release from the endoplasmic reticulum. The Ca2+ sensitivity of these channels allows internal Ca2+ to regulate the electrical activity of the cell membrane. Activating these potassium channels controls many physiological processes, from the firing properties of neurons to the control of transmitter release. This review will discuss what is understood about the Ca2+ sensitivity of the two best-studied groups of Ca2+-sensitive potassium channels: large-conductance Ca2+-activated K+ channels, KCa1.1, and small/intermediate-conductance Ca2+-activated K+ channels, KCa2.x/KCa3.1.The Ca The α subunits of these channels comprise six transmembrane helices (S1–S6), the pore region between helices S5 and S6), and cytosolic N- and C-terminal domains. Their biophysical characteristics are their independence from membrane voltage because they have lost most of the positively charged residues commonly associated with voltage-dependent gating [2+ concentrations activate KCa2.x channels through a unique calmodulin (CaM) gating mechanism.  and gain-of-function (GOF) variants in the corresponding genes, respectively, underlies a broad spectrum of human channelopathies [Ca2.1 channels in humans. While neurodevelopmental disorders such as cerebellar ataxia and tremor are associated with loss-of-function KCa2.2 mutations [Ca2.3 are linked with the Zimmermann–Laband syndrome (ZLS) [KCNN3 was linked to schizophrenia. On the other hand, another study [Ca2.3 channel gene (L283fs287X) results in the deletion of the protein N-terminal region identified in schizophrenic patients. These resulting mutant KCa2.3 channels were found to dominantly suppress KCa2.3 channel currents [Ca3.1 are linked with a subset of hereditary xerocytosis (HX) (OMIM 194380), which is an autosomal dominant congenital hemolytic anemia characterized by erythrocyte dehydration [The discovery of mutations in genes encoding Kr organs . Decreasopathies ,61. Therutations . The GOFme (ZLS) ,62,63,64me (ZLS) ,62,65, Ime (ZLS) , CAG reper study  found thcurrents   and respond rapidly to changes in Ca2+ with time constants of 5–15 ms [Cahe cells ,10. The  5–15 ms .2+-binding protein calmodulin (CaM) serves as the KCa2.x and KCa3.1 channel’s Ca2+ sensor [Ca1.1 channels are both Ca2+ and voltage-activated, and their sensitivity to voltage and intracellular Ca2+ are prominently influenced by their association with auxiliary and non-pore-forming modulatory β (β1-4), γ (γ1-4), and LINGO1 subunits [Ca1.1 channel’s Ca2+ binding sites. The KCa1.1 structure  simulations. The observations determined that mutations that decrease hydrophobicity at the HA-S4–S5 interface led to Ca2+ hyposensitivity of KCa2.x channels. The mutation that increases hydrophobicity results in hypersensitivity to Ca2+ [2+ sensitivity of the amino acid sequences of KCa2.x, which disclosed several differences between their channel subtypes. More studies are needed for the potential drug development for channelopathy disorders.Recently, the Cryo-electron-microscopy structures of the entire K channel  have sig to Ca2+   [Ca3.1 mutations are linked with hereditary xerocytosis (HX) [Genetic mutations encoding K tremors ,77. Gainme (ZLS) ,79 and ime (ZLS) . In contsis (HX) .KCNMA1-linked channelopathy mutations are primarily associated with neurological conditions, including movement disorders, seizures, and intellectual disability. These mutations comprise GOF and LOF alterations in KCa1.1 channel activity and some variants of unknown significance (VUS). Over the past decade, a great effort has been devoted to the Ca2+ sensing mechanisms of Ca2+-activated K+ channels. Each channel type uses a unique molecular mechanism to regulate its Ca2+ gating. Site-directed mutagenesis, in silico modeling, and electrophysiology have proven to make powerful tools in studying Ca2+-dependent gating that leads to the development of more selective biophysical and pharmacological approaches for the KCa channelopathy.Human Ca2.x channel’s apparent Ca2+ sensitivity.The hydrophobic interactions between the HA helix and S4–S5 linker regulate the KCa channels reviewed as Sensitive K


This ion regulates a plethora of cellular functions ranging from fertilisation and birth to development and cell death. A sophisticated system is responsible for maintaining the essential, tight concentration of calcium within cells. Intricate components of this Ca2+ network are store-operated calcium channels in the cells’ membrane. The best-characterised store-operated channel is the Ca2+ release-activated Ca2+ (CRAC) channel. Currents through CRAC channels are critically dependent on the correct function of two proteins: STIM1 and Orai1. A disruption of the precise mechanism of Ca2+ entry through CRAC channels can lead to defects and in turn to severe impacts on our health. Mutations in either STIM1 or Orai1 proteins can have consequences on our immune cells, the cardiac and nervous system, the hormonal balance, muscle function, and many more. There is solid evidence that altered Ca2+ signalling through CRAC channels is involved in the hallmarks of cancer development: uncontrolled cell growth, resistance to cell death, migration, invasion, and metastasis. In this work we highlight the importance of Ca2+ and its role in human health and disease with focus on CRAC channels.All human life starts with a calcium (Ca Thus, it is important to know—all human life starts with a calcium wave.Society’s relationship with natural science is at a critical level. Never have the issues affecting science been more demanding and challenging, the public more interested, or the opportunities more apparent. However, public confidence in scientific advice has been shaken within the last 2 years of the COVID-19 pandemic. The progress of biotechnology seems to be far ahead of the awareness and approval of many people. We are now in a climate of deep concern due to public mistrust, unease, and occasional hostility. Since the start of the prevailing pandemic in 2020, science has made immense progress, especially in the design of a new era of vaccines. Unfortunately, to date, scientific progress and its knowledge are still out of reach for the majority of people. This leads to unwarranted doubts as well as disbelief in science and scientific facts. We believe this is primarily due to a misunderstanding of science amongst the lay population, especially in a time when more and more scientific publications are becoming freely accessible. In our opinion, the scientific community is missing interpersonal and clear communication skills. With this review, we thus emphasise communication of our scientific knowledge on Ca2+) is a major element of human life. It is the first signal in a fertilised egg, which occurs when the envelope of a female oocyte is breached by a male sperm cell [2+ is absorbed mainly through dairy products and green leafy vegetables [2+ as a vital mineral is quantitatively the most important in the human body. The main amount (roughly 99%) is stored in bones and teeth, but the Ca2+ ion fulfils far more important roles in humans. Ca2+ serves a courier role inside our cells , meaning its concentration within cells is changed in response to an outside (extracellular) signal. This extracellular signal is called “first messenger” or “ligand”. Once its messenger-function is activated, Ca2+ holds remarkable roles important for fertility, birth, development, muscle contraction, and the function and regulation of cells and tissues; it plays important roles within our immune system and is associated with viral life cycles [Calcium . A permanent increase of the Ca2+ concentration inside the cell (within the cytosol) is toxic and can lead to processes where our immune system attacks the body’s own cells , inflammation of tissues, and other pathophysiological  conditions [2+ levels at all times. This monitoring system includes diverse transporters and ion channels within our cells to keep the concentration of Ca2+ in a temporally defined and tight range. Upon activation by a ligand, the cytosolic concentration of Ca2+ increases by 1000-fold through the release of Ca2+ from stores of the endoplasmic reticulum (ER—a network used to transport various substances around the cell). To maintain the high level of Ca2+, which is needed for cellular functions, the entry of extracellular Ca2+ is obligatory [2+ concentration can be increased in two different ways: either by releasing Ca2+ from ER stores within the cell, or by allowing Ca2+ entry through the cell membrane   . As the 2+ can enter the cell via Ca2+ ion channels embedded in the plasma membrane. A primary route for this Ca2+ influx is through “store-operated channels”. These types of channels open in response to a signal of empty internal Ca2+ stores. This implies that there must be a communicated trigger signal from the cells’ interior to open up the “gate” in the membrane. The prototypic store-operated channel fulfilling these criteria is the so-called Ca2+ release-activated Ca2+ (CRAC) channel. In the normal physiological state, the CRAC channel transports Ca2+ into the cell just when needed. However, if this channel is changed in any way  this can have serious impacts on our health.As mentioned above, Ca2+ concentration within. This protein is called stromal interaction molecule 1 (STIM1) [2+ channel [The origin of all proteins lies in our genetic code, the deoxyribonucleic acid (DNA), which we inherit from our parents. Proteins are built according to instructions stored in certain areas of our DNA (the “genes”), which are essential for our survival. The CRAC channel system is based on the interaction of two proteins: one of them is partially located inside the ER, able to sense the Ca (STIM1) ,15. The  channel ,17  to bridge the distance to the usually far apart plasma membrane, allowing it to directly interact with Orai1 in the created puncta [2+ ions to enter the cell and refill the internal stores.STIM1 traverses the membrane of the endoplasmic reticulum and includes a Cad puncta . Once th2+, which is not able to enter the cells due to the erroneous Orai1 channel, culminating in severe immune impairment. Physiologically healthy Orai1 proteins associate in groups of six, forming a so-called “hexamer”. This bunch of six proteins assembles in a circle with a hole in the centre that represents the actual funnel for Ca2+ entry  . There, 2+ entry . The str opening . In the 2+ signal, have been associated with a variety of human diseases, underscoring their importance.Such a two-component system as it exists for the CRAC channel is highly unusual in biology as it involves proteins from two different cellular compartments, the ER and PM, respectively. However, it is precisely this evolutionary feat that makes this system so important for humans. To date, mutated STIM1 and Orai1 proteins, and therefore an altered Ca2+ signalling is highly coordinated in space and time. One can easily believe that a disruption of these precise mechanisms can lead to defects [2+ entry in patients with a severe form of immunodeficiency  were reported to result in fatal infections with bacterial and viral pathogens [2+ into the cell when needed. This phenomenon, also called channelopathy , has serious implications for our immune cells: without proper Ca2+ homeostasis, they simply are paralysed. The occurring syndrome in patients is characterised by recurrent and chronic infections, immune cells attacking our own cells (“autoimmunity”), a lower-than-normal number of platelets in the blood (“thrombocytopenia”), a state of low muscle tone (“muscular hypotonia”), problems with the skin , and the inability to sweat (“anhidrosis”) [The complex pattern of Ca defects ,23. The athogens ,25,26,27athogens ,17,28. Bdrosis”) . To examine pathological functions of genes in living organisms, scientists usually make use of model organisms to study specific mutations in proteins. For an animal to become a model organism in science, it must have several characteristics: a comparatively small size and it should be easy to keep and reproduce quickly. Only under these conditions can scientific results be obtained with reasonable effort and in a relatively short time. Mice usually serve as prototypic model systems—hardly any mammalian organism has been studied as intensely as the mouse—its genome  has been completely decoded and 99% of the genes in the mouse genome have a similar form in humans . The use2+ entry into the cell and patients suffer from an excessive contraction of the pupil of the eye (“miosis”), thrombocytopenia, intellectual disability, muscle fatigue, the absence of normal spleen function (“asplenia”), and they exhibit a condition where the skin becomes dry and horny (“ichthyosis”). A related disease is tubular aggregate myopathy , a similar multisystemic disease, varying in disease severity and age of onset [Apart from LoF mutations that can occur in proteins, alterations with the consequence of higher activity of the protein may also lead to disease. In this regard, a gain-of-function (GoF) mutation in STIM1 is associated with the so-called Stormorken syndrome (named after its discoverer) . This Goof onset ,38,39. Vof onset , a syste2+—a task accomplished by STIM and Orai proteins. An essential role of CRAC channel proteins has also been proven for cells of the innate and adaptive immune system in humans ,42. The 2+ is also well-established to be important for the transmission of signals via nerve cells (neurons) and for the contraction of heart muscles (cardiomyocytes). There is evidence that Ca2+ is involved in neurodegenerative diseases such as Alzheimer’s, Huntington’s, and Parkinson’s disease [2+ signals are furthermore known to play an essential role in infection with diverse viruses. Therefore, Ca2+ is important for virus-host cell interaction [2+ dynamics within a cell [2+ can bind to severe acute respiratory syndrome coronavirus 2, the COVID-19-causing virus (SARS-CoV-2), leading to a facilitated viral entry into human cells [2+ channels a feasible drug target site to prevent COVID-19; this has already been successfully proven to be effective against the common flu (influenza A) or ebolavirus-triggered disease [2+-dependent mechanisms of SARS-CoV-2 have been fully deciphered and are still under investigation [Ca disease . In addi disease . Ca2+ sieraction , viral reraction ,9,43. Vin a cell . During an cells ,46. This2+ homeostasis are also linked to cancer. Cancer as the leading cause of death globally comprises more than 100 different types, affecting any part of the body in both sexes [2+: (i) enhanced growth (“proliferation”) of cells , (ii) resistance to programmed cell death , and (iii) migration, invasion, and metastasis  of cancer cells [2+ channel blockers were identified as inhibitors of cancer growth [2+ activates several key signalling pathways and transcription factors (needed for the production of proteins) that control cell proliferation and the cell cycle in cancer cells [Apart from viral infections, disturbances in Cath sexes . Cancer th sexes . Pathophth sexes ,50. Of ter cells ,52,53. Oer cells ,55. The r growth ,57,58. Mer cells ,60,61,622+ entry is through store-operated Ca2+ channels, especially CRAC channels [2+ is essential for human brain tumour glioblastoma invasion [2+ homeostasis leads to resistance to apoptosis [Most major carcinomas develop from epithelial origin, meaning cells that line hollow organs and glands. In epithelial cells, the main pathway for Cachannels ,64. To wchannels . Furtherinvasion  and for invasion ,67,68. Minvasion ,70. Moreinvasion ,72. It hpoptosis ,74, due poptosis ,77,78,792+ is low inside the ER, but they differ at key sites that interact with STIM1. STIM2 is more sensitive to Ca2+ but is weaker in activating Orai. Not all tissues and cell types need all of the homologs, implying that there are differences in the requirement of their expression. Yet the interplay of STIM1 and STIM2 with the Orai proteins shapes the Ca2+ signal and is important in deciding for a specific cellular response and not the other. Moreover, STIM2 and Orai2/3 are relevant to cancerous processes, whereby Orai2 was, for instance, reported to promote migration and metastasis in gastric cancer [Unmentioned so far, there are other very similar proteins besides Orai1 and STIM1. The information for the synthesis of further Orai and STIM proteins is encoded within the DNA of humans; these are referred to as Orai2, Orai3, and STIM2. These “relatives” are scientifically termed “homologs” and, much like siblings, show common and differentiating features and functions. All three Orai proteins can, for instance, be activated by STIM1 if Cac cancer ,81 and ic cancer . STIM2 ic cancer , while Oc cancer  and is lc cancer . Overall2+ plays a major role in human health and disease. To date, there are no drugs currently being used clinically that target the Ca2+ signalling machinery, although several pharmaceutical companies are working on it. In order to develop new drugs, the molecular structures of the proteins involved must first be deciphered [2+ signalling machinery in cells. Understanding the structure of CRAC channels, meaning STIM and Orai proteins, will improve the prospects for developing novel and relatively untapped therapeutic agents which aim at combating the growing list of human diseases associated with aberrant store-operated Ca2+ influx in humans, including cancer and COVID-19.As outlined in this work, Caciphered . This isCommunicating the current state of science to society is often a difficult task. We hope this paper is a first step in helping people without a background in biology understand the important role calcium plays in humans and the importance of research being conducted on this topic. No one benefits from scientific progress if it is not understood and broadly accepted.


Our research has shown evidence of oxidative stress, mitochondrial dysfunction, an elevated expression of Ca2+ entry channels, Ca2+-dependent pumps and exchangers, and an abnormal rise in cytosolic Ca2+ in human glaucomatous LC fibroblast cells. We have evidence that this increase is dependent on Ca2+ entry channels located in the plasma membrane, and its release is from internal stores in the endoplasmic reticulum (ER), as well as from the mitochondria. Here, we summarize some of the molecular Ca2+-dependent mechanisms related to this abnormal Ca2+-signalling in human glaucoma LC cells, with a view toward identifying potential therapeutic targets for ongoing optic neuropathy.Glaucoma is one of the most common causes of treatable visual impairment in the developed world, affecting approximately 64 million people worldwide, some of whom will be bilaterally blind from irreversible optic nerve damage. The optic nerve head is a key site of damage in glaucoma where there is fibrosis of the connective tissue in the lamina cribrosa (LC) extracellular matrix. As a ubiquitous second messenger, calcium (Ca Glaucoma is the one of most common causes of treatable visual impairment in the developed world , affectiThe LC’s topography in glaucoma includes shearing and a collapse of its beams, resulting in a thinning and backward bowing of the LC. Histologically, there are significant changes in the extra-cellular matrix (ECM) in human and monkey glaucoma optic nerve head specimens , with inThe lamina cribrosa cells of the ONH were first characterized by the Hernandez group in 1988 . FurtherThese LC cells stain positively for α-smooth muscle actin (α-SMA), fibronectin, collagen1A1, and vitronectin, and they appear to occur in humans (and likely in other primates) but have not been identified in rodents such as mice or rats. These latter animals undergo a gliosis at the optic nerve head  .The cells that play a role in this CT remodeling include ONH astrocytes and LC cells. Hernandez has shown a significant number of alterations in LC astrocytes, including an increase in the synthesis of ECM macromolecules, cell adhesion molecules and growth factors, and cytokines . Fibrosis is attributed to excessive ECM accumulation which ultimately damages the connective tissues . Externa2+ signalling, and mitochondrial dysfunction in glaucoma LC cells.It has been found that the CT of the LC and the trabecular meshwork (TM) show substantial ECM fibrosis in glaucoma ,18. Our 2+) were abnormally elevated in glaucoma LC cells i levels found in glaucoma LC cells i) results from either the influx of extracellular Ca2+ through the plasma membrane Ca2+ entry channels or its release from internal stores such as the endo/sarcoplasmic reticulum, primarily through 1,4,5-trphosphate receptor (IP3R) and ryanodine receptors (RyR). In most cells, external stimuli bind to ligand-engaged G protein-coupled receptors (GPCRs), leading to the subsequent synthesis of IP3 and the activation of the IP3 receptor at the ER membrane, resulting in the release of Ca2+ from the ER i at the lower levels so that small increases in plasma membrane Ca2+ influx or efflux from the internal store can trigger rapid, transient, and marked increases in [Ca2+]i. It is these increases in [Ca2+]i that are a key signal in gene transcription regulation. Carocesses ,35. It im the ER ,37. In rchondria ,39. The the cell . Thus, c2+ plays a key role in multiple signal transduction pathways in a wide variety of cell types by modulating critical cellular functions such as cell death and gene transcription. For example, the pro-hypertrophic ECM gene expression in cardiac myocytes is seen in pathological cardiac growth and is characterized by the elevation of cytosolic Ca2+, acting via numerous signalling cascades, including the protein kinase c α (PKCα) pathways i also induced a sequential phosphorylation of PKCα and the downstream series of phosphorylation of p38MAPK and p42/44 MAPK, resulting in Ca2+-dependent genes, such as profibrotic ECM genes, and, ultimately, the proliferation of glaucoma LC cells i in response to changes in membrane potential . It. It2+ repoptosis ,91. 2+ is a major player in the regulation of cell death i are known to play a pivotal role throughout the cell cycle [2+ [2+ is required early in G1, as cells re-enter the cell cycle, to promote the activation of AP1 (FOS and JUN) transcription factors, c-AMP-responsive element binding (CREB) protein, and NFAT. Ca2+ plays a key role through these factors in coordinating the expression of cell cycle regulators such as the D-type cyclins, which are required for the activation of cyclin-dependent kinase 4 complexes. The initiation of the centrosomal duplication at the G1/S phase is also dependent on Ca2+ and on calmodulin (CaM), CaM kinase II (CaMK), and the centrosomal protein CP110. The Ca2+/CaM/CaMK pathways were shown to be necessary for cell cycle progression [2+-dependent phosphatase, is known to activate the nuclear factor of the activated T-cell transcription factor NFAT, and a demonstrated link with MYC [2+-dependent pathways and proliferation.It is well-known that cell proliferation is dependent on the cell cycle, which consists of four primary phases: G1, the first phase; S phase, in which nucleic acids occurs; G2, the second phase; and M phase, or mitosis. The switches between these phases are tightly controlled, and checkpoints during the cell cycle determine if the cell proceeds to the next phase . These cll cycle . Severalycle [2+ ,118. Ca2gression ,118. Calwith MYC  regulati2+/calcineurin/NFAT pathway is one of the major routes that can be activated by the entry of Ca2+ through plasma membrane Ca2+ channels. The use of various Ca2+ channel blockers has supported the idea that Ca2+ influx plays a role in cell proliferation. These observations suggest that a decrease in Ca2+ channel expression will lead to cell cycle arrest [2+ signalling involved in cell proliferation. It has been found that the enhanced TRPC3-dependent Ca2+ influx led to increased proliferation in ovarian cancer [The Cae arrest . Howevern cancer . 2+ entry channels TRPC1/C6 contribute to oxidative stress-induced ECM gene transcription and cell proliferation [In human LC cells, we previously found that the Caferation . The TRPferation . Furtherferation   in adenocarcinoma cells [2+ concentration, such as vitamin D3, ATP, ionomycin, and thapsigargin (an inhibitor of the ER Ca2+-ATPase pump), induced autophagic cell death in MCF-7 breast cancer cells [2+-dependent induction of autophagy, Ca2+ released from intracellular stores or fluxed from extracellular space via distinct Ca2+ channels activate CaMKKβ, which mediates the AMPK-dependent inhibition of mTORC1 [It is known that ER stress-induced autophagy depends on Ca-7 cells . Other sma cells . Furtherer cells . In the f mTORC1 . StudiesStudies on human primary cultures of TM cells have also shown that during glaucoma, the autophagic mechanism in TM cells is dysregulated. TM cells isolated from glaucomatous patients show dysregulation in the autophagic signalling pathway and a reduction in the autophagic response to oxidative stress . The samIn human LC cells, we found that glaucoma LC cells exhibit a significant increased number of peri-nuclear lysosomes and an increase in autophagy in glaucoma LC myofibroblasts . Glaucom2+ homeostasis is a crucial determinant of cellular function and survival. Intracellular Ca2+ ions are dynamically regulated by the plasma membrane, endoplasmic reticulum, and mitochondria. Ca2+ is also a ubiquitous and versatile intracellular second messenger contributing to several critical signalling pathways that participate in the regulation of numerous physiological processes [2+ enters into a cell and interacts with different Ca2+-binding proteins, of which there are over 200 encoded by the human genome that function either as Ca2+ effectors or buffers. Disruption of cytosolic Ca2+ homeostasis/signalling is responsible for many pathological states including proliferation, apoptosis, autophagy, angiogenesis, fibrosis, neurodegeneration, and cancer diseases. This review highlights the multiple abnormalities that we have identified in Ca2+ homeostasis in glaucomatous lamina cribrosa cells associated with increased fibrosis in the optic nerve head (summarized in Carocesses ,40. In r


Changes in Ca2+ through altered calcium channel expression or activity are known to promote tumorigenesis and treatment resistance in a range of cancers including BC. Emerging evidence shows that this is mediated by Ca2+ modulation, supporting the function of tumour suppressor genes (TSGs) and oncogenes. This review provides insight into the underlying alterations in calcium signalling and how it plays a key role in promoting disease progression and therapy resistance in TNBC which harbours mutations in tumour protein p53 (TP53) and the PI3K/AKT pathway.Triple-negative breast cancer (TNBC) is characterized as an aggressive form of breast cancer (BC) associated with poor patient outcomes. For the majority of patients, there is a lack of approved targeted therapies. Therefore, chemotherapy remains a key treatment option for these patients, but significant issues around acquired resistance limit its efficacy. Thus, TNBC has an unmet need for new targeted personalized medicine approaches. Calcium (Ca Triple-negative BC (TNBC) accounts for ~15% of all BC cases and is characterized by a clinically more aggressive disease, which is linked with higher recurrence rates, increased metastatic potential and poorer overall survival-4. Unlike oestrogen receptor-positive (ER+) BC and human epidermal growth factor receptor 2 (HER2)-positive BC, TNBC lacks the expression of oestrogen, progesterone and HER2 receptors,6. In both ER+ and HER2-positive BC, the expression at the protein and/or gene level of these receptors has been successfully targeted as a cancer treatment with either small molecule inhibitors or monoclonal antibodies. The results of these interventions in ER+ and HER2-positive BC have resulted in both improved response rates and overall survival of those BC patients,9. In TNBC, the development of targeted approaches to treat the disease lags behind other BC subtypes meaning that TNBC has one of the poorest survival rates of all breast cancer subtypes,11.Breast cancer (BC) is the most common female-specific cancer in the world. In recent years, advances in treating TNBC patients with novel targeted agents have shown benefits. For example, for patients who have a germline breast cancer gene (BRCA) 1/2 mutation, poly(ADP-Ribose) polymerase (PARP) inhibitors are used to treat TNBC patients in both the adjuvant and metastatic settings-15. However, patients with BRCA mutations account for only a small percentage of TNBC cases. Immunotherapy offers new hope for TNBC patients: results from the recent IMPASSION study demonstrate that immunotherapy combined with chemotherapy offers benefit to a subset of patients who have elevated programmed death ligand (PDL) 1 expression. Lastly, tumour-associated calcium signal transducer 2 (TROP2), encoded by the (TACSTD2) gene, is a transmembrane glycoprotein expressed in approximately 80% of TNBC. The antibody-drug conjugate (ADC) sacituzumab govitecan, a TROP2-directed antibody and topoisomerase inhibitor drug conjugate, has been approved for the treatment of TNBC by the FDA. However, there remains a significant proportion of TNBC patients for whom these therapies offer little benefit.In TNBC, chemotherapy remains one of the main treatment options for patients, but acquired resistance to chemotherapy remains a significant clinical problem2+) is an essential component required for normal cellular function and is involved in the regulation of processes such as metabolism, muscle contraction and phagocytosis as well as cell growth, proliferation and apoptosis,19. Local Ca2+ concentrations oscillate with varying frequency and amplitude, enabling the induction or modulation of signal transduction and gene transcription-22. In line with this, it has become evident that Ca2+ supports the functions of key tumour suppressor genes (TSGs) and oncogenes commonly altered in BC, such as tumour protein p53 (TP53), phosphatase and tensin homolog (PTEN) and Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), as well as a number of others reviewed elsewhere. Generally, oncogenes promote cellular survival by dampening Ca2+ signalling, whereas TSGs induce apoptosis through Ca2+ influx. Owing to this key role, it is unsurprising that Ca2+ is altered downstream of key driver genes, enabling cancer progression and treatment resistance,24,25. Consequently, Ca2+ represents an area of interest for the development of new drug therapies targeted to specific genomic alternations-28. This review outlines in detail the mechanisms of Ca2+ modulation and how these mechanisms are altered in specific TNBC cohorts who harbour mutations in TSGs such as TP53 and PTEN and oncogenes such as PIK3CA.Calcium , as well as long-term genomic changes by regulating signal transduction and gene transcription. In non-excitable cells such as epithelial cells in BC, Cai2+ is predominately modulated through store-operated calcium (SOC). Here, store-operated calcium channels (SOCCs) allow the influx of Ca2+ into the cytosol due to differential Ca2+ concentrations which are established and tightly regulated through the continual action of various pumps and calcium channels,24,29. Extracellular Ca2+ is maintained at a concentration of 2 mM and cytosolic Ca2+ at a concentration of 100 nM. In addition, the ER and mitochondria act as Ca2+ stores, holding Ca2+ concentrations of ~1 mM and ~200 nM, respectively. These differential calcium gradients allow for localised changes in Ca2+ through the action of calcium channels, which in turn promotes various functions such as signal transduction and gene transcription as well as cellular processes such as proliferation and apoptosis.Ca2+ release from the endoplasmic reticulum (ER) through associated calcium channels such as inositol trisphosphate (IP3) receptors (IP3Rs) or ryanodine receptors (RyRs). IP3R activation is initiated by upstream activation of plasma membrane G protein-coupled receptors (GPCRs) and receptor tyrosine kinase (RTK). Following activation of these receptors by their associated ligands, phospholipase C (PLC) enzyme is produced, hydrolysing phosphatidylinositol 4,5-bisphosphate (PIP2) and resulting in the production of diacylglycerol (DAG) and IP3, the latter of which activates IP3R inducing ER Ca2+ release. In addition, the channel can also be activated by common cellular stresses found in the tumour microenvironment (TME), such as reactive oxygen species (ROS), ER stress, altered cellular energetics, hypoxia and drug treatment. Alternatively, RyR releases Ca2+ from the ER upon sensing changes in intracellular Ca2+. Subsequently, the decrease in store Ca2+ is detected by a family of stromal interaction molecule (STIM) channels, which mediate store-operated calcium entry (SOCE) primarily through calcium release-activated calcium modulator (ORAI) and occasionally transient receptor potential (TRP) channels at the plasma membrane-38. Normal Ca2+ concentrations are subsequently restored through Ca2+ efflux via the plasma membrane calcium pump (PMCA) and Na+/Ca2+ exchanger (NCX) along with reabsorption into the ER through the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA). In addition, it has been increasingly found that the membrane potential of cancer cells has become more depolarised, and as such voltage-gated calcium channels (VGCC) have also been shown to contribute to calcium entry,40. Furthermore, these channels have also been shown to contribute to SOCE in cancer cells.SOC is initiated by Cai2+ is a common feature in cancer that is able to promote neoplastic transformation and drug resistance,42,43. This is mediated through altered calcium channel expression or activity, enabling cancer hallmarks such as migration/invasion, proliferation, survival and apoptotic resistance,27,28,44. Typically, increased Cai2+ enhances proliferation, while a reduction and faster recovery are linked to apoptotic resistance and decreased sensitivity to chemotherapeutic agents. To date, several studies have shown various calcium channel families are altered in BC and linked to tumorigenesis. Importantly, this work has also uncovered specific differences in channel expression between BC subtypes. For example, IP3R2 and IP3R3 have been found to be upregulated in TNBC tissue compared to luminal subtypes. SOCE is also altered in TNBC through increased ORAI1 and STIM1 expression, which promotes invasion and migration, while in patient samples, it was linked to poorer prognosis,48. In contrast, ORAI3 was found upregulated in luminal and HER2 BC. SOCE inhibitors in early preclinical studies have demonstrated an ability to inhibit proliferation and migration in MDA-MB-231 TNBC cell lines and reduce tumour growth in TNBC mouse models. In addition, Azimi et al. (2017) observed that TRPC1 is significantly increased in TNBC compared to all other BC subtypes, resulting in an enhanced epithelial-mesenchymal transition (EMT) phenotype. Going forward, this review focuses on the role of Ca2+ in supporting TSGs and oncogenes commonly altered in TNBC; however, altered calcium channel expression in BC subtypes has been extensively reviewed elsewhere,46,52.Research has continued to demonstrate that aberrant Ca,53-55. This heterogeneity partly explains the limited impact that targeted therapeutic approaches have made in treating the majority of TNBC patients. Part of this heterogeneity is associated with the mutational background of TNBC, which could impact a patient’s response to therapeutic intervention. However, this could provide opportunities for scientists and clinicians to design novel approaches to treat TNBC.TNBC is a heterogeneous disease, which includes distinct molecular subtypes,57. Normally, TP53 is activated due to various stress signals such as DNA damage, hypoxia, ROS, oncogenic activation and cancer treatment. In this way, TP53 helps prevent tumorigenesis by regulating biological processes such as apoptosis, cell cycle, DNA repair and senescence. Mutations that result in loss of TP53 activity and impair its normal cellular functions have been linked to chemoresistance as well as reduced overall survival-64. Most (90%) functionally relevant TP53 mutations produce missense products that have both loss- and gain-of-function features,66 and are more resilient to degradation. Most known cancer-associated TP53 coding DNA sequence (CDS) changes are within the DNA-binding domain corresponding to exons 5-8 and amino acids 102-292, with > 28% of these nonsynonymous SNPs occurring at eight key sites. Many of these originate from aberrant CpG methylation. Notably, recurrent TP53 aberrations during cancer growth result in selective sweeps of new clones with independent mutant TP53 proteins.Somatic mutations in the TSG TP53 (located at 17p13.1) occur in ~80% of TNBC cases2+, TP53 is known to have a non-transcriptional role in the cytosol, inducing apoptosis in cells by regulating Ca2+ release from the ER,70. Recent research has shown that wild-type TP53 localises to ER and mitochondria-associated membranes (MAMs), where it interacts with SERCA increasing Ca2+ loading in the ER by enhancing its activity,72 [2+ transfer to the mitochondria inducing pro-apoptotic mitochondrial overload, leading to the release of pro-apoptotic factors.In relation to Caivity,72 . In addi,72,73. Here, TP53 fails to induce SERCA activity, reducing mitochondrial Ca2+ response, which also reduces caspase 3 and PARP cleavage, thus promoting an anti-apoptotic phenotype [et al. (2015) demonstrated that wild-type TP53 cells were sensitive to doxorubicin (Adriamycin) treatment, as TP53 mediated an increase in cleavage of PARP and caspase 3, which resulted in reduced cell survival. However, in TNBC TP53 mutant MDA-MB 468 cell lines, doxorubicin failed to increase both SERCA activity and Ca2+ levels, thus conferring chemotherapy resistance. This critical role of TP53 in promoting pro-apoptotic Ca2+ in treatment sensitivity was further confirmed by Giorgi et al., who demonstrated that overexpression of SERCA or mitochondrial calcium uniporter (MCU) restored treatment sensitivity. This effect was lost when Ca2+ levels were reduced by chelation, highlighting how Ca2+ is integral to the function of TP53. In addition, mouse xenograft tumours established using mouse embryo fibroblast (MEF) H-ras induced cells were observed to double in size with TP53 loss compared to wild-type controls, corresponding with an observed decrease in Cai2+ Activity.Mutant TP53 disrupts these processes, leading to apoptotic resistance and reduced sensitivity to chemotherapyhenotype . In addii2+ changes in BC. TRP channels promote calcium entry at the plasma membrane following activation by PLC, DAG or store release from IP3R channels. These channels support store refilling via SOCE through the interaction with ORAI and STIM. A novel anti-neoplastic organic derivative GaQ3  was demonstrated to induce apoptosis by promoting TP53 expression through elevated Cai2+ concentrations,78. The key role of Ca2+ in this mechanism was confirmed using the Ca2+ quencher TMB-8, which prevented an increase in Cai2+, leading to an inhibition of TP53-mediated apoptosis. Further work found that this increase in Cai2+ was mediated by the binding of TP53 to the TRPC6 promoter, resulting in the overexpression of TRPC6, enhancing Ca2+-dependent apoptosis in MCF-7 BC cells. A putative TP53 binding site on the TRPC6 promoter was also identified by bioinformatic analysis using the Mat Inspector module of the genomatix database, suggesting TP53 is a potential regulator of TRPC6. Thus, it was identified that the Cai2+ released through TRPC6 is mediated by TP53 and plays a major role in promoting apoptosis in a range of cancer cell line models including MCF-7,78.Other calcium channels such as TRP have also been linked to TP53 pro-apoptotic Ca,79. A recent study demonstrated through bioinformatic analysis that ORAI3 was elevated in patients with poor response or residual disease following chemotherapy treatment, which was also predictive of poor patient outcomes. Increases in ORAI3 expression induced resistance to the chemotherapeutic drugs cisplatin, 5-FU and paclitaxel by reducing apoptosis and increasing survival of T47D and MCF7 BC cell lines. These effects were found to be mediated by ORAI3, promoting a decrease in TP53 and cyclin-dependent kinase inhibitor 1A (p21). This was confirmed by the removal of extracellular Ca2+ and/or Orai3 functionality, which resulted in an increase in TP53 expression and reduced chemoresistance. Interestingly, Hasna et al. (2018) also discovered that the Phosphoinositide 3-Kinase (PI3K)-Protein Kinase B (AKT) pathway was also induced by the observed increase of ORAI3 expression in chemoresistant cells. PI3K inhibitors partially inhibited resistance induced by TP53 expression. This chemoresistant effect was mediated by PI3K induction of the SGK-1, leading to TP53 degradation via MDM2 proto-oncogene (Mdm2) and Nedd4-like E3 ubiquitin-protein ligase (Nedd4-2). Prior to this work, Brickley et al. (2013) highlighted a link between SOCE and SGK-1 in TNBC cell lines. SOCE activation induced the expression of SGK-1 following exposure to cellular stress. This mechanism appeared cytoprotective, as siRNA targeting SGK-1 under the same conditions increased apoptosis.Research in a range of BC subtypes including TNBC has also observed that altered SOC channel expression is linked to apoptotic and chemotherapy resistance through the TP53 pathways. Specifically, the expression of S100P is elevated in TNBC and linked to chemotherapy resistance and poor survival-86. Gibadulinova et al. identified reduced phosphorylation and activity of TP53 in response to DNA damage following S100P binding in a range of cancer cell lines including BC cell lines MCF7 and T47D. When bound to TP53 protein, S100P promotes cell survival and resistance towards anticancer drugs such as paclitaxel and cisplatin, evading senescence and thus promoting cancer progression.Proteins such as S100P are also linked to TP53, where their expression was found to be altered in BC, enabling drug resistance. This is primarily because TP53 expression is lost in ~10% of cases, and in the other cases, the function of the mutant protein is changed. However, advances in drug design have enabled both scientists and clinicians to develop drugs such as Eprenetapopt (APR-246) and COTI-2, which aim to reactivate TP53. These TP53 reactivators have been tested in preclinical studies in TNBC but also in clinical trials of TP53-mutant myelodysplastic syndromes (MDS) and solid tumours (NCT02433626). Further investigation is warranted to ascertain if these drugs impact any of the calcium signalling mechanisms mentioned previously.Until recently, it has been challenging to develop drugs that can target TP53 for cancer treatment2+-dependent TP53 apoptosis and associated mechanisms contribute to poor outcomes in TNBC and resistance to chemotherapy. Consequently, it highlights calcium channels as a therapeutic target to modulate Ca2+ concentrations in mutant TP53 TNBC, which, through TP53 restoration or calcium channel activation drugs, could lead to improved responses to existing treatments and better overall patient survival.Taken together, there is compelling evidence that Ca. Alterations in this pathway are common in TNBC and are associated with poorer outcomes and treatment resistance. Up to 30% of TNBC tumours harbour aberrations in the oncogenic PI3K/AKT pathway through mutation of either AKT serine/threonine kinase 1 (AKT1) or PIK3CA,94. The loss of wild-type TP53 regulation of AKT (located at 14q32.33), PIK3CA (at 3q26.32) and PTEN (at 10q23.31) has consequences for the PI3K-AKT pathway. PIK3CA encodes an alpha subunit of Phosphatidylinositol 3-Kinase (PI3K). PTEN is another TSG whose mutation can mimic the effects of mutant TP53. PTEN is a negative regulator of the PI3K/AKT pathway through downregulation of phosphorylated AKT, and it is also commonly altered in TNBC,96.PI3K/AKT oncogenic signalling through pathway activation is linked to cell survival, growth and apoptosis. Mutations at these four genes may be oncogenic on their own, or may occur in tandem with other TSG mutations: PIK3CA, PIK3R1 (encoding regulatory protein p85α) and PTEN commonly co-occur in BC. Changes at AKT, PIK3CA and PTEN are much less common than those at TP53. PIK3CA mutations are typically missense and constitute ~10%-18% of cases, and in ~4% of cases, PIK3R1 may be mutated,100 - often, PIK3CA and PIK3R1 co-occur independently. PTEN is mutated in ~6%-7% of cases and AKT1 is rarer at ~3%. Although all 4 genes may have cancer-related SNPs, TP53 tends not to have other mutation types, in contrast to AKT and PIK3CA that can be amplified or possess CNVs; additionally, PTEN tends to be inactivated, often by deletion.TP53, AKT, PIK3CA and PTEN changes together constitute the genetic basis of the majority of TNBC cases and initiate the initial stages of tumour development. mTORC2 activates AKT, thus leading to cell proliferation and survival. mTORC1 is activated by phosphorylated AKT and promotes protein translation and cell growth,104. TP53 has three primary domains - transactivation domain, DNA-binding domain and tetramerization domain. TP53 is regulated by the MDM2 proto-oncogene (Hdm2) protein via binding and physical separation from target genes, resulting in low levels of TP53 in normal cells. TP53 regulates PI3K via binding and inhibition of PIK3CA, which encodes p110α, and reduces AKT activation. Mutations in TP53 may lead to hyperactivation of PIK3CA and the PI3K signalling pathway. Studies have demonstrated the regulatory effects of TP53 on PTEN via the functional TP53 binding site present within the PTEN promoter region, resulting in inhibition of PtdInsphosphorylation and subsequent AKT activation. AKT can enhance the function of Hdm2 via phosphorylation, and evidence suggests that PTEN may prevent the degradation of TP53 through suppression of AKT activation. PTEN has also been identified as a stabiliser of TP53 by way of physical association between the two, conferring protection from degradation to TP53. Despite these stabilising functions, deletion of PTEN and subsequent activation of the PI3K pathway have been shown to activate TP53, with significantly increased TP53 expression present in PTEN-/- cells.There is an established link between the PI3K/AKT pathway and TP53 . RTKs ar.PTEN has also been identified as a stabiliser of TP53 by way of physical association between the two, conferring protection from degradation to TP53. Despite these stabilising functions, deletion of PTEN and subsequent activation of the PI3K pathway has been shown to activate TP53, with significantly increased TP53 expression present in PTEN-/- cells. A study investigating the mechanisms involved in the regulation of PIK3CA transcription via TP53 in ovarian cancer described two promoters present on the PIK3CA gene that have been shown to directly bind TP53 resulting in transcriptional inhibition. It was found that suppression of TP53 resulted in increased levels of p110α transcripts, proteins and PI3K signalling pathway activity, while overexpression of TP53 resulted in decreased levels of p110α protein. Loss-of-function mutations in TP53 correlate with hyperactivation of PIK3CA and the PI3K pathway, resulting in worse prognoses for head and neck squamous cell carcinoma patients.TP53 can regulate cell survival via inhibition of the PIK3CA as well as the PI3K/AKT pathway, independent of PTEN. This transcriptional downregulation of PIK3CA via TP53 has been demonstrated in head and neck cancer cell lines, where the PTEN protein was not detected and induction of TP53 resulted in decreased PIK3CA expression and reduced AKT phosphorylation, indicating the transcriptional regulatory effects of TP53 on PIK3CA2+ release from ER stores via interaction with IP3R [2+ oscillations to either promote cell survival or induce apoptosis. Under hypoxia conditions or chemotherapy treatment, IP3R promotes Ca2+ transfer to the mitochondria, inducing the release of pro-apoptotic factors. However, the anti-apoptotic protein AKT has been shown to phosphorylate IP3R, preventing ER Ca2+ transfer to the mitochondria and inhibiting apoptosis,111 [,111. The PI3K/AKT pathway is also negatively regulated by the TSG PTEN, which is commonly lost or mutated in TNBC and associated with poorer prognosis as well as treatment resistance,114. PTEN is known to localise at the ER and reduce AKT-induced phosphorylation of IP3R, thus promoting ER Ca2+ release. In addition, it also associates with MAM and regulates the transfer of Ca2+ to the mitochondria, enabling Ca2+-mediated apoptosis. In PTEN mutant BC, IP3R becomes phosphorylated and inactivated by AKT, decreasing Ca2+ release from the ER and associated transients to the mitochondria, reducing the sensitivity to Ca2+-mediated apoptosis. This work highlights a potential link between PTEN loss and AKT activation, supporting treatment resistance and disease progression via deactivated IP3R mediated apoptotic resistance.Research has demonstrated that the PI3K/AKT pathway can modulate Caith IP3R . This caosis,111 . This, i2+ release from ER can be finetuned through interaction with calcium binding proteins such as neuronal calcium sensor-1 (NCS-1). Recent studies in TNBC cells have shown that overexpression of NCS-1 increases Ca2+, promoting survival. Furthermore, this mechanism was linked to paclitaxel resistance, where NCS-1 binding to IP3R promotes drug resistance,118 [. Furthermore, Singh et al. (2017), analysing MCF-7 cells, demonstrated that IP3R inhibition is able to induce autophagy and subsequent cell death, an effect not observed in non-tumorigenic cells. In addition, IP3Rs are also associated with the nuclear envelope and nucleoplasmic reticulum, where I3PRs can also regulate Ca2+ levels within the nucleus to promote cell proliferation,121. In TNBC, reducing nuclear IP3R Ca2+ by chelation decreased cell proliferation and induced tumour necrosis. Overall, this research identified that IP3R can impact both PI3K/AKT dependant and independent pathways.In relation to IP3R, its ability to mediate Caance,118 . Interes2+ store release via IP3R, which is modulated by PI3K-AKT/PTEN pathways, has an indirect contribution to drug-resistant mechanisms by association with Ca2+ binding proteins. For example, Ca2+/calmodulin-dependent kinase (CAMKK) is essential for AMP-activated protein kinase (AMPK) induction following activation by epidermal growth factor (EGF) in TNBC cell lines. AMPK is an essential regulator of AKT activation under cellular stress such as drug treatment and hypoxia. Ca2+ is a requirement for this mechanism as its chelation using BAPTA-2AM completely inhibits AMPK-induced phosphorylated AKT. Specifically, AMPK phosphorylates s-phase kinase-associated protein 2 (Skp2) at S256, triggering the formation of an E3 ligase complex that promotes activation of AKT. This outlined mechanism was shown to promote cell survival and apoptotic resistance. In BC patient samples, Skp2 activation correlates with increased AKT and AMPK expression and is associated with poor survival outcomes. In addition, Han et al. (2018) found that targeting AMPK-Skp2 in TNBC MDA-MB-231 cell lines increased their sensitivity to anti-EGF receptor-targeted therapy. Furthermore, this study also showed in the same TNBC cell lines that this mechanism is not only induced by EGF but also hypoxia, promoting survival under such conditions. Hypoxia is a common characteristic in the tumour microenvironment (TME), which is also linked to chemotherapeutic resistance.Ca. Using TNBC MDA-MB-231 cell lines, Bhattacharya et al. further demonstrated that (in the presence of phosphorylated AKT) ORAI3- and STIM1-mediated Ca2+ promotes the induction of Snail family transcriptional repressor (SNAIL). Induction of SNAIL expression is known to mediate EMT, a cellular phenotype linked to drug resistance,127. In addition, TRPC1 has also been shown to play a role in EMT of TNBC via regulation of hypoxia-inducible factor 1 alpha (HIF-1α) and AKT signalling in PTEN-deficient BC cells. Azimi et al.  also noted that TRPC1 has a prognostic value in basal BC a molecular subtype associated with TNBC. Specifically, high TRPC1 expression was associated with significantly poorer patient relapse-free survival. Furthermore, it was noted that TRPC1 can induce EMT and promote chemotherapy resistance in MDA-MB-468 TNBC cell lines through the upregulated ATP-binding cassette, subfamily C, member 3 (ABCC3), a multidrug resistance ATP-binding cassette (ABC) transporter. In addition, this channel, alongside ORAI1 and IP3R2, has been found to be upregulated following doxorubicin treatment in MDA-MB-231 TNBC cells.SOCE mediated through ORAI and STIM as well as TRP enables the refiling of ER stores following PI3K-IP3R induced release, which supports signal transduction. Both ORAI and STIM have been shown to be upregulated in TNBC, enabling disease progression and promoting treatment resistance. Adriamycin (ADM/doxorubicin) was found to induce an increase in Cai2+ which was reduced with siRNA targeting of TRPC5. Exploration of the underlying pathway discovered that Ca2+ mediated through TRPC5 could activate CaMKKβ, AMPKα and mammalian target of rapamycin (mTOR), which resulted in the induction of autophagy. As noted above, AMPK modulates AKT activation, which can regulate ER store release, and this can then facilitate chemotherapy resistance. In the study by Zhang et al. (2017), targeting the TRPC5-mediated CaMKKβ/AMPKα/mTOR pathway increased sensitivity to ADM. Furthermore, analysis of patient samples via a tissue microarray (TMA) found that TRPC5 was significantly increased in patient samples post chemotherapy treatment when compared to pre-treated samples. Supporting the role of TRPC5 in chemotherapy resistance, studies have linked TRPC5 expression to the induction of expression of the multidrug resistance protein 1 (MDR1) in TNBC cells.TRPC5 is another family member whose overexpression is linked to chemotherapy resistance by induction of a protective autophagy mechanism which promotes cancer cell survival,132 and AKT inhibitors such as capivasertib and ipatasertib have been conducted in this setting. Interestingly, these trials have reported clinical activity and benefits to patients. The identification of key biomarkers of response to these treatments and their study in combination with chemotherapy or other targeted therapies warrants further investigation.Because of the large number of TNBCs that have an activated PI3K/AKT pathway, it is unsurprising that early phase clinical trials of both PI3K inhibitors such as buparlisib2+ signalling and those TNBC cancers that have a TP53 mutation and/or activation of the PI3K/AKT pathway. Activation of the PI3K/AKT pathway can be achieved through either mutation of PIK3CA/AKT1 or loss of expression of PTEN. Our review highlights that several calcium channels associated with Ca2+ release from ER and subsequent SOCE are linked to the activation of the PI3K/AKT pathway. Aberrations in the PI3K/AKT pathway disturb Ca2+ handling and enable escape mechanisms such as cell survival and apoptotic resistance, thus promoting chemotherapy resistance.Furthermore, as discussed above, there is a strong link between Ca-28,135. Several calcium blocking drugs have been developed which can modulate calcium channels and be exploited as cancer treatments,135,136. Calcium channels provide a plethora of targets to modulate Cai2+, and ion channel targeting drugs are currently the second largest group of FDA-approved drugs, therefore providing a bank of new drugs which can be repurposed for the treatment of cancer.The role of calcium channels in mediating therapy resistance through interplay with genomic alternations in TNBC highlights calcium channels as potential targets for personalized medicine approaches. The study of calcium channel blockers (CCBs) is an area of intense research2+ signalling, promote Ca2+-calmodulin kinase (CamK) activity and reverse the suppression of TSGs such as secreted frizzled related protein 1 (SFRP1), tissue inhibitor of metallopeptidase-3 (TIMP-3) and WNT inhibitory factor 1 (WIF-1), leading to cell death of colorectal cells. Other similar studies identified the ability of the immunosuppressant drugs to regulate SOCE, namely, leflunomide used to treat arthritis and teriflunomide used for multiple sclerosis. In addition, the drug eflornithine, also known as DFMO, has been found to reduce SOCE and promote a decrease in cell proliferation and cell death resistance in colorectal cancer. Eflornithine has been found to inhibit TRPC1 expression, which, as outlined above, plays a role in AKT signalling in PTEN deficient TNBC.Several studies have identified existing FDA-approved drugs that can modulate store-operated release and entry through key channels such as ORAI, STIM, TRP and IP3R, as outlined herein, thus representing potential future therapeutics for the tackling disease progression and chemotherapy resistance in the TNBC cohorts outlined. For example, an extensive screen of 1118 unique FDA-approved drugs discovered 11, mainly cardiac glycosides, which have the ability to modify Ca. The metabolite of aspirin, salicylate, has been found to inhibit SOCE and mediate mitochondrial uncoupling, reducing mitochondrial Ca2+ uptake and leading to a decrease in the proliferation of colon cancer cells. Furthermore, other NSAIDs such as ibuprofen and indomethacin have also been shown to modulate SOCE through STIM1, which decreased colorectal cancer growth. The CCB mibefradil, previously used to treat hypertension, can also inhibit SOCE by blocking ORAI channels, promoting cell apoptosis and cell cycle arrest. In addition, a potent synthetic oestrogen used in the treatment of prostate and breast cancer, diethylstilbesterol, can inhibit SOCE via TRPM7 channels,145. The potential to target SOCE modulation for disease benefit has led to the development of a number of drugs, such as CM2489, which has entered into phase 1 clinical trial for psoriasis; if successful, it could prove useful for TNBC treatment. CM2489 is a first-in-class calcium release-activated calcium (CRAC) channel inhibitor that targets ORAI channels. Lastly, IP3R channels appear to be a key target in PI3K/AKT pathways mediating chemoresistance; both caffeine and heparin have been shown to inhibit these channels and could hold future potential as novel treatments in combination with existing anti-cancer agents,148.Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly prescribed anti-inflammatory, anti-pyretic drugs that have demonstrated anti-cancer activityi2+ mediated through calcium channels and how this plays an important role in promoting disease progression and therapy resistance in TNBC harbouring mutations in key TSG and oncogenes. Specifically, TNBC with mutant TP53 is associated with a loss of Ca2+ store release via SECRA, leading to reduced mitochondrial Ca2+ loading; this, in turn, promotes cell survival and apoptotic resistance, thus enabling treatment resistance. In addition, I3PR-mediated ER store release and SOCE modulation are altered by PI3K/AKT activation, promoting apoptotic resistance. Consequently, the outlined evidence highlights calcium channels as therapeutic targets to modulate altered Cai2+ downstream of common genomic alterations in TNBC.TNBC is characterized as an aggressive form of BC associated with poor patient outcomes. For the majority of patients, there is a lack of approved targeted therapies. TNBC has frequent genomic alternations in TP53 and the PI3K/AKT pathway. This review provides insight into the underlying alterations in Ca2+ signalling. It may be that targeting Ca2+ signalling, whilst beneficial for one cancer, could be detrimental for another. Overall, this review highlights Ca2+ electroporation as an emerging area in TNBC that could hold significant potential as disease biomarkers as well as future therapeutics when combined with treatments that can inhibit the PI3K-AKT pathway or reactivate TP53 expression.This promise is supported by the fact that a growing number of calcium targeting drugs are under development, as well as a number of existing FDA-approved drugs. However, owing to the complexity of calcium signalling, its ubiquitous nature and differential expression between cell types and genomic alterations, care needs to be exercised when targeting Ca