The study of immune evasion has gained a well-deserved eminence in cancer research by successfully developing a new class of therapeutics, immune checkpoint inhibitors, such as pembrolizumab and nivolumab, anti-PD-1 antibodies. By aiming at the immune checkpoint blockade (ICB), these new therapeutics have advanced cancer treatment with notable increases in overall survival and tumor remission. However, recent reports reveal that 40–60% of patients fail to benefit from ICB therapy due to acquired resistance or tumor relapse. This resistance may stem from increased expression of co-inhibitory immune checkpoints or alterations in the tumor microenvironment that promotes immune suppression. Because these mechanisms are poorly elucidated, the transcription factors that regulate immune checkpoints, known as “master regulators”, have garnered interest. These include AP-1, IRF-1, MYC, and STAT3, which are known to regulate PD/PD-L1 and CTLA-4. Identifying these and other potential master regulators as putative therapeutic targets or biomarkers can be facilitated by mining cancer literature, public datasets, and cancer genomics resources. In this review, we describe recent advances in master regulator identification and characterization of the mechanisms underlying immune checkpoints regulation, and discuss how these master regulators of immune checkpoint molecular expression can be targeted as a form of auxiliary therapeutic strategy to complement traditional immunotherapy. Cancer is a multi-faceted disease that while complex to treat possesses multiple vulnerabilities which can be targeted therapeutically. Of the several hallmarks of cancer, the study of tumor evasion has garnered interest in unraveling underlying mechanisms and identifying therapeutic targets. In recent years, there has been a rapid development of immunotherapy strategies; for example, synthetically derived chimeric antigen receptor (CAR) T cells which recognize and direct specific cytotoxicity to target cells, recombinant cancer vaccines that prompt the immune response against tumors, and immune checkpoint blockade (ICB) therapy which inhibits cancer immune evasion .Immune checkpoint (IC) inhibitors have earned well-deserved appreciation for their efficacy and efficiency in combating cancer immune evasion . IC inhiAs the ICB resistant patient population expands, there is an increased need to overcome resistance mechanisms or circumvent it through modified forms of therapy. Much of current research is focused on either downstream effectors or upstream regulators (considered as “master regulators”) to modulate response to drug treatment as a promising avenue to overcome the resistance. This concept has been successfully applied to identify ERK and FOXM1 as master regulators of cell fate and proliferation, respectively ,18. As sTo shed light on ICB therapy’s limitations, we must first review briefly the interactions between tumor and immune cells. The immune response is highly regulated yet flexible and adaptive to protect the host against a myriad of pathogens. This process relies on recognizing pathogens with high sensitivity while preventing autoimmunity . However+ T cell infiltration during the active response, CD8+ T cells were abundantly present and restricted at the tumor margin, suggesting T cell-induced cytotoxicity was no longer effective [The molecular mechanisms underlying ICB resistance have not been fully understood. However, it is generally accepted that ICB resistance is dynamic interplay between cancer cells, T cells, and tumor microenvironment (TME). One such manner in which ICB resistance is developed is by the insufficient tumor-recognizing T cells generation. This occurs by genetic or epigenetic alterations that govern the formation, presentation, and processing of tumor neoantigens, or foreign proteins unique to tumors. Changes in the signaling cascade that regulate cytotoxic T cell activity also provides an advantage to cancer cells to resist immune recognition and modulate the microenvironment to favor tumor growth and progression ,24. Anotffective . This maffective . Consequffective ,15.Another limitation of ICB therapy is the growing number of immune-related adverse events (irAEs). Immune checkpoint inhibitors have a different spectrum of toxicities when compared to chemotherapeutic or other biological agents, and the severity of irAEs appears to depend on immunotherapy regimens. For instance, in a phase III clinical trial that tested the safety and efficacy of atezolizumab and bevacizumab (anti-VEGF-A) plus carboplatin and paclitaxel (chemotherapeutic agents) in non-small lung cancer (NSCLC) resulted in pneumonitis ,29,30. TDrosophila melanogaster [Susumu Ohno’s term, “master regulator” is defined as “a gene that occupies the very top of a regulatory hierarchy” and is not under the regulatory influence of any other gene. This term was coined to describe the regulatory mechanism of sex determination in nogaster . Since inogaster , and “a nogaster , and celnogaster  because nogaster . Since tSince master regulators hold such a pivotal role in regulating gene expression of numerous targets and thereby have implications in tumorigenesis, much effort has been focused on identifying such regulators in various cancers. In the context of ICB, this has stimulated the search for potential regulators of immune evasion mechanisms. As stated earlier, the process of immune evasion uses multiple pathways, including the modulation of TME, to generate an immunosuppressive and pro-tumoral environment. Hence, the master regulators identified for facilitating this process would have regulatory roles in multiple immune cells and may have varied functions depending on each cell type.+ T cells and regulator T cells (Tregs), inhibiting the maturation of immature dendritic cells, and enhancing the production of ROS in Myeloid-Derived Suppressor Cells (MDSCs) [Consider the example of STAT3, a known master regulator of cancer immunity , as show (MDSCs) . STAT3 i+ T cells further the immunosuppressive environment by limiting its accumulation in tumors. This is carried out by inhibiting the production or activation of CXCL10 in tumor-associated myeloid cells. In one study carried out to assess the function of STAT3 on CD8+ T cells, it was seen that removal of STAT3 results in an increased expression of CXCL10 receptor (CXCR3), which increases an accumulation of T-cells at the tumor site [For instance, IL-10 and TGF-β mediate STAT3 activation and induce FOXP3 expression, which yields a positive feedback loop for the increased production of those cytokines and functions to maintain the inhibitory phenotype Tregs . These cmor site .Hence, with the presence and activation of STAT3, we can infer that it holds a negative regulatory role in releasing cytokines that draw affinity towards tumor infiltration. Lastly, STAT3 is one of the main transcription factors that govern MDSC functions to promote tumor proliferation and suppress immune-mediated cytotoxic cell death of cancers . MDSCs eRecently, it was found that the same regulators of cell survival possess control over the expression of ICs, namely AP-1 , MYC 5555, and EAnother form of regulation by these master regulators is to enhance anticancer immunity by suppressing the expression of co-inhibitory ICs or promoting the expression of co-stimulatory ICs. This phenomenon is reflected in the negative regulation of PD-L1 by ER-α signaling in breast cancer. ER-α regulates gene transcription through the direct binding of the receptor to regulatory regions (enhancers or silencers) of target genes . The relWe summarized the list of known master regulators that elicited regulatory functions on the ICs expression, together with their broader roles in the context of oncoimmunology, in To define the master regulators as the transcriptional regulators located at the hub of the gene regulatory network, one could apply a systematic identification, wherein the master regulator should be enriched for a list of differentially expressed genes (DEGs) of a particular phenotype of interest . For exaWhile the discovery of master regulators does provide important insight into biological functions, to translate these findings in a clinical setting, therapeutic strategies must be devised. In the growing interest of master regulator discovery, several reports present candidate master regulators as potential therapeutic targets. This encourages further studies to investigate the therapeutic outcome of targeting these master regulators. Several strategies have been developed to harness the therapeutic potential of master regulators, including;Modulating the expression of these master regulators to sensitize cancers to other treatmentsUsing in silico databases to find candidate small molecules to reverse signaturesDirect inhibition or stimulation of transcription factorsIKZF1, leading to an enhanced immune infiltrate recruitment and tumor sensitivity to ICB therapy. Chen JC, et al., identified the master regulator IKZF1 that was sufficient to induce the recruitment of immune infiltrates and immune-mediated cytotoxicity in the autoimmune disease [An example of the first strategy is seen in a study that enhances the master regulator  disease , which t disease . This stAs for the second strategy, a number of methods and resources have been designed to assist the identification of master regulators for a given phenotype and the enrichment of small molecules that reverse the gene expression signature. This is illustrated in the integrated transcriptomics study done in lung adenocarcinoma to identify master regulators using the ARACNe algorithm and reposition drug candidates to revert the pathological gene expression profile using the MRCMap pipeline . De BastThe third strategy, wherein the master regulators are targeted directly, has reached the early phase clinical trial (NCT02716012). MTL-CEBPA is the first-in-class small activating RNA oligonucleotide drug that targets the master regulator CCAAT/enhancer-binding protein alpha (C/EBP-α) for hepatic and myeloid functions and multiple oncogenic processes ,104,105.http://ilincs.org), an integrative web-based platform for the analysis of omics data and signatures of cellular perturbations stored in the Library of Integrated Network-based Cellular Signatures (LINCS) [We anticipate that implementing strategies as aforementioned to harness the therapeutic potential of master regulators may draw out the unforeseen limitations. Considering that this paradigm would be gaining traction in oncoimmunology research, there is a lack of prior studies regarding master regulators of ICs to reference and provide a foundation for new investigations. However, this knowledge gap also presents an opportunity for investigators to venture into and bridge the gap. Another drawback of adopting this new concept is the lack of available therapeutics that directly inhibit master regulators of ICs. This limitation may impede investigations from holistically perceiving the full effect master regulators have in cancer progression and encumbers translating their findings in a clinical setting. However, mechanisms can still be tested using gene manipulation techniques such as CRISPR, non-coding RNAs, and/or gene overexpression to prove the concepts, encouraging researchers and pharmaceutical companies to design therapeutics for these candidate targets. Rapid growing in the field of computational drug discovery, artificial intelligence, and big pharmacogenomic/proteomic databases and tools ,114,115  (LINCS) ,112,113.This review summarized the current limitations of ICB therapy and how it can be potentially circumvented by identifying and altering master regulators. The oncoimmunological functions of known master regulators are reviewed with emphasis on how these factors may regulate immune checkpoint molecule expression. While still a niche subject in oncoimmunology research, targeting the master regulators of ICs holds promise in becoming the new arm of precision immunotherapy. We further discuss possible approaches for developing therapeutic strategies around the master regulators.


Non-shivering thermogenesis in mammalian brown adipose tissue is a powerful mechanism to defend normothermia in cold climates. To minimize the loss of chemical energy, the central functional component, mitochondrial uncoupling protein 1, UCP1, must be tightly regulated. The canonical pathway of UCP1 activation includes lipolytic release of free fatty acids in response to an adrenergic signal. Activating fatty acids overcome constitutive inhibition of UCP1 by the di- and triphosphate forms of purine nucleotides, i.e., ATP, ADP, GTP, and GDP. Cellular concentrations of inhibitory, free nucleotides are subject to significant, adrenergically induced alterations. The regulatory components involved may constitute novel drug targets to manipulate brown fat thermogenesis and thereby organismic energy balance. We here review evidence for and against a dominant role of nucleotides in thermogenic control, describe conceptual routes to endogenously and pharmacologically alter free nucleotide pool size, speculate on a signaling role of degradation products released from active brown fat, and highlight gaps in our understanding of signaling and metabolic pathways involved. Brown adipose tissue (BAT) provides a mechanism for adaptive, non-shivering thermogenesis to endotherm mammals [reviewed in ]. IsolatFor decades, the divergent roles of actively controlled free fatty acid levels as UCP1 activators and passively acting nucleotides as constitutively present inhibitors remained the widely accepted model of thermogenic regulation in BAT. The arguments seemed obvious enough: millimolar cytosolic concentration of ATP alone seemed sufficient to fully and efficiently shut down UCP1 function at all times, exceeding the nanomolar or low micromolar dissociation constant by several orders of magnitude –13. To aRemaining doubts were convincingly countered and brought to the point in a publication by Klingenberg, in which he finds that “the conclusion that nucleotides do not play a role in intracellular regulation of UCP1 because of a too high affinity, seems to be unfounded” . ArgumenIf active regulation of free nucleotides is assumed to be implicated in the activation of UCP1 mediated non-shivering thermogenesis, brown adipocytes require a mechanism to reduce their concentration in response to an adrenergic stimulus, either by a shift in the balance between free and complexed nucleotides or by an altered overall amount. There is evidence for both.-adrenergic stimulation of brown adipocytes  expression is strongly and very quickly induced in BAT by cold exposure, both on the transcript and the protein level , 32. At SE119452 ]. These SE119452 . Indeed,SE119452 ; Figure In summary, even under control conditions, inhibitory purine nucleotide concentrations in the cytosol of brown adipocytes are in the range of their UCP1 dissociation constant. Adrenergic stimulation of non-shivering thermogenesis decreases both total pool size by enzymatic degradation and the fraction of inhibitory free nucleotides by changes in calcium concentration and pH. Activation of UCP1 includes the concerted action of both free fatty acid liberating and nucleotide degrading processes.in vivo remains to be studied. If so, inhibitors of human brown fat thermogenesis may be considered for the treatment of cachexia, a progressive body mass loss in cancer patients recently linked to brown fat energy wasting (Adrenergic stimulation leads to degradation of purine nucleotides in brown adipocytes contributing to the activation of UCP1 mediated thermogenesis. Enzymes involved in purine metabolism are targets of a large number of approved and experimental drugs for indications as diverse as gout , viral  wasting –40.Therapeutic targeting of human brown fat thermogenesis, however, is more often discussed in the context of metabolic disease and envisions activation, not inhibition, of the immense oxidative capacity of this tissue. At least young adults feature an amount of brown adipose tissue that, fully activated, would cause a sizeable negative shift in energy balance , potentiAdrenergic stimulation leads to purine nucleotide loss in brown adipocytes. Mere changes in phosphorylation state between ADP/ATP and GDP/GTP can bi-directionally be caused by a large variety of enzymes and pathways. Similarly, the monophosphate forms AMP and GMP are readily formed or converted back to diphosphates by adenylate and guanylate kinases. Immediately upon thermogenic activation, formation of the second messengers cAMP and cGMP may, to some extent, contribute to a reduction in ATP and GTP concentration before being eliminated to AMP and GMP . Since cGSE119452 (Since full activation of UCP1 mediated thermogenesis and changes in nucleotide concentration occur rapidly upon adrenergic stimulation , 48, 49,SE119452 ]. SimilaSE119452 . In striSE119452 ]. While More promisingly, facultative binding of AMPD to the plasma membrane strongly impedes catalytic activity , a phenoThe significance of purine nucleotides in the control of non-shivering thermogenesis was one of the first milestones in the mechanistic understanding of brown adipose tissue mitochondria, predating the identification of their target, uncoupling protein 1. As a means of acute thermogenic activation, nucleotides have long been neglected as passive inhibitors being displaced by actively regulated free fatty acid levels. In fact, cytosolic free purine nucleotide concentrations are adrenergically modified by several routes acting in concert, including calcium complex formation and enzymatic nucleotide degradation. The signaling cascade connecting norepinephrine and purine metabolic enzymes remains unresolved, and at least in the case of adenine nucleotides, plausibly relies on α1-adrenoreceptors and phospholipase C. Beyond the diligence work of elaborating these unknown pathways, the release of degradation products by active brown adipocytes presents the fascinating opportunity to discover novel markers and endogenous indicators of non-shivering thermogenesis . While tAB-H and TF wrote this review article in concert. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


RNA-protein complexes (RNPs) are essential components in a variety of cellular processes, and oftentimes exhibit complex structures and show mechanisms that are highly dynamic in conformation and structure. However, biochemical and structural biology approaches are mostly not able to fully elucidate the structurally and especially conformationally dynamic and heterogeneous nature of these RNPs, to which end single molecule Förster resonance energy transfer (smFRET) spectroscopy can be harnessed to fill this gap. Here we summarize the advantages of strategic smFRET studies to investigate RNP dynamics, complemented by structural and biochemical data. Focusing on recent smFRET studies of three essential biological systems, we demonstrate that investigation of RNPs on a single molecule level can answer important functional questions that remained elusive with structural or biochemical approaches alone: The complex structural rearrangements throughout the splicing cycle, unwinding dynamics of the G-quadruplex (G4) helicase RHAU, and aspects in telomere maintenance regulation and synthesis. Many cellular processes rely on the faithful interaction between proteins and RNA, which constitute most major molecular machineries. For some of these RNA-protein complexes (RNPs), structural data has been available for quite some time, the most prominent example being the ribosome . However0), leading to high FRET efficiencies when the two fluorophores come into closer proximity. This allows applying FRET spectroscopy as a molecular ruler, covering distances ranging from approximately 2 to 10 nm. First FRET experiments were performed in an ensemble approach, therefore averaging over all molecules in potentially different conformations. However, applications in a single molecule setup allow observation of individual, non-synchronized molecules with different conformations and dynamics. Detection in single molecule resolution is achieved by applying total internal reflection fluorescence (TIRF) microscopy or confocal based setups. However, TIRF based approaches are the most widely used weapon of choice. This technique requires immobilization of the FRET-labeled constructs to the imaged surface. Independent of the imaging technique, strategic construct design is a crucial step to achieve significant results using smFRET. Labeling sites are chosen based on structural data (if available) corresponding to an expected high distance variance upon conformational changes preferably crossing the Förster radius. In studies on RNP dynamics, RNA or proteins can be targeted for fluorophore labeling [FRET is based on the non-radiative energy transfer from a donor to an acceptor fluorophore that show a spectral overlap of emission and absorption spectra, respectively. This transfer is distance dependent to the sixth power and scales with the FRET pair specific Förster radius  and a varying number (~100) of proteins. The recent advances in structural biology have yielded the first models of the spliceosome core structure ,27,28, aact-complex), (5) formation of the catalytic core by U2 and U6, (6) transesterification and formation of a lariat structure (C-complex), (7) additional remodeling steps, and (8) second transesterification and release of the ligated exons.Several crucial steps can be defined that are required for efficient splicing: (1) Recognition of the 5′ splice site (5′SS), which occurs by binding of the pre-mRNA to the U1 RNA (commitment complex 2), (2) Binding of U2 RNA to the branch site (A-complex), (3) Recruitment of the trimeric snRNP (tri-snRNP) into the complex (B-complex), (4) multiple conformational rearrangement and remodeling steps, during which U1 and U4 are removed from the complex  assists Prp5 in prespliceosome activation and confers ATP dependency of the remodeling process . It has 2+ analysis of their RNA construct showed coexistence of a high FRET state of 0.96 corresponding to IIc, a FRET efficiency of 0.54 for IIa and an additional, minor low FRET conformation of 0.35, all three dynamically interconverting into each other  accumulation, the authors could characterize the clade deriving from the commitment complex 2, the A-complex, the BIn summary, the studies presented here demonstrated how conformational analysis of parts of the spliceosome afford a functional connection between individual components of this highly complex RNP. The level of complexity requires both of the approaches presented here: bottom-up, where individual (sub-) domains of RNAs and proteins are put into focus, and top-down, where a less well-defined splicing activity conferred by cellular extracts are analyzed to yield insights from both directions.The key structural element of the complex protecting the telomeres are intricate DNA structures consisting of three guanosine tetrads, termed G-quadruplexes (G4s) . These sThe structure of these G-quadruplex elements depends on various factors, such as sequence composition, loop size, the cation bound into the quadruplex core, and the number of molecules involved in formation of quadruplex structures, i.e., inter- or intramolecular structures ,49,52. IAside from their function within telomeres, these structures have been described to be functionally relevant in processes like transcriptional pausing, replication, translation, and RNA processing. They exert their effect by utilizing the intrinsic thermal stability, leading to steric interference, competition with other conformations, or as recruiting platform for other proteins .The broad spectrum of structures G4s are able to adopt, and their broad variety of binding partners as well as their intrinsic dynamics render these structures extremely difficult to study in their functional context. It is therefore not surprising that studies focusing on G4 structures in complex with e.g., protein binding partners are scarce.There have been a number of proteins described that are able to unfold G4 structures in DNA as well as in RNA . The mosRecent research on the RNA G4 helicase RHAU is focusing on the binding mode and unwinding mechanism on intramolecular DNA and RNA G4s. However, structural data obtained from crystal structures and small angle X-ray scattering (SAXS) even in combination was not sufficient to characterize the unwinding process, as it provided only snapshots. The authors therefore opted to complement their structures of different complex compositions  by employing smFRET to investigate the conformationally dynamic nature of the unwinding process.Previous structural and biochemical data revealed selective binding of RHAU to parallel DNA as well as RNA G4 ,60. smFRDrosophila. In general, there are a number of differences described between bovine and Drosophila RHAU, showing why a detailed analysis at the level of individual molecules is of advantage.Moreover, a single stranded overhang of at least 9 nucleotides is required for efficient RHAU binding to G4s ,57. LateThe co-crystal structure of the DEAH-box helicase RHAU and a G4 DNA individually describes the importance of several protein domains  and partial disruption (EFRET 0.4) of the G4, no FRET fluctuations between the two states were detected, which indicates that there is no refolding step in the absence of ATP. Upon addition of ATP, refolding of the G4 by RHAU was observed in three distinct steps. The last step of refolding was followed by a rapid decrease back to a FRET state at 0.4, which was assigned to be a second single-step unfolding , which also contains termini of the linear DNA at each end. In order to prevent undesired recombination or other erroneous processing of these DNA ends (as they may mistakenly be recognized as double-strand breaks), complexes termed shelterin are placed onto and shield each of the chromosome ends . These pTelomerase holoenzyme RNP complexes are assembled and processed in the nucleus and nucleolus before being selectively localized at chromosome ends . The useThe unique architecture and functionality of telomerase enzymes has made a detailed functional characterization as well as structural biology approaches difficult for a long time. Initial, partial structures of protein domains from diverse organisms including the reverse transcriptase TERT ,66, acceTogether with a number of single molecule-based approaches, the structures also shed light on aspects of telomerase subunit folding ,75,76,77Especially for the human telomerase holoenzyme however, the overall arrangement of the RNA component relative to TERT was difficult to establish. One particular obstacle was the discontinuous set of structural and functional elements within hTR see .In order to elucidate the orientation of these domains towards each other as well as upon binding to TERT, Parks et al. combined an extensive set of smFRET experiments with a modeling approach . They plTo efficiently immobilize fully reconstituted telomerase holoenzyme species, a specific primer substrate showing high affinity towards the assembled complex was used . In abseThe additional information derived in this study was however obtained by including the distance restraints derived from FRET experiments into a structural model of hTR architecture that was in line with previous experimental datasets ,79. ConsWhile telomerase provides one factor to extend the successively shortened G-rich strand, the C-rich complementary strand also has to be selectively synthesized . In ordeHow the CST complex is able to make the G-rich strand accessible for further DNA synthesis is less well understood. Here, an approach combining biochemical and smFRET analysis of DNA duplexes with different overhang lengths and positions in presence of the CST complex provided deeper insight : In elecBinding of CST in situ caused an immediate shift into a low-FRET, G4 unfolded conformation B, and a These findings however were derived from the CST trimer in absence of in-depth functional knowledge about any of the proteins. These individual contributions were then addressed in a follow-up study , showingTo test how these different observations were exerted by the different subunits of the CST complex, the authors expressed and purified “CS” and “ST” complexes containing Ctc1-Stn1 and Stn1-Ten1, respectively. They assessed the G4 unfolding activity of these complexes in smFRET experiments and compared them to the results using the full CST complex . They foThis again provides an elegant example how carefully crafted smFRET experiments are able to fill in gaps in the functional understanding of such processes by comparative analysis of subcomplexes, and the combination of such experiments with biochemical assessment of substrate recognition.Taken together, the studies on various RNP complexes presented here all clearly show that strategic smFRET experiments can significantly improve the understanding of complex systems, on their conformationally dynamic nature but just as much on their structural composition.The research on the spliceosome summarized here shows how smFRET was able to shed light onto the complex rearrangements necessary throughout the splicing cycle, where structure determination such as crystallization and cryo-EM only delivered snapshots. With different labeling schemes on either protein, snRNA, or substrate RNA it was possible to detect the RNA- and ATP-dependent closing of Prp5, the spontaneous or assisted conformational toggling of U2 snRNA in dependence of ions, proteins and modification as well as pre-mRNA dynamics throughout the whole splicing cycle. While some of these principles have been hypothesized previously, single-molecule data could provide crucial evidence for their existence. Furthermore, these smFRET studies were able to additionally find and visualize novel intermediates and pathways.The presented studies on G4 unwinding by RHAU show the benefit of single molecule experiments when combining them with structural data on RHAU. The identification of distinct states throughout the unwinding process, the dynamics that come along with both the interaction with the enzyme and the ATP hydrolysis step and, finally, the impact of mutations on such dynamics all would have been extremely difficult to rationalize from structural or biochemical data alone.The unique telomerase mechanism proved to be especially challenging to decipher. The smFRET data gave significantly deeper insights into many of the aspects in telomerase folding, assembly and activity, and demonstrated how questions that were again difficult to tackle by structural biology means were more readily amenable by single molecule techniques. Telomerase holoenzyme structures published in the last years confirmed that several previously hypothesized insights into the mechanism of telomerase-catalyzed DNA synthesis were correct. This mostly concerned the binding locations of proteins and their interactions with the RNA, but also the general architecture and arrangement of the RNA within the telomerase holoenzyme. They also further structurally explained how the telomerase cycles during DNA synthesis, combining nucleotide addition processivity and repeat addition processivity to efficiently add significant amounts of DNA onto the chromosome ends.By looking at strategically reconstituted subcomplexes, it was again the view into individual molecules that precisely identified functional roles for each protein within the CST complex.In summary, the work showcased here provides a number of prominent examples in how functional questions on RNP complexes can be tackled by combining and comparing structural biology techniques with FRET spectroscopy on the level of individual molecules.


The intrinsically disordered proteins belonging to the LATE EMBRYOGENESIS ABUNDANT protein (LEAP) family have been ascribed a protective function over an array of intracellular components. We focus on how LEAPs may protect a stress-susceptible proteome. These examples include instances of LEAPs providing a shield molecule function, possibly by instigating liquid-liquid phase separations. Some LEAPs bind directly to their client proteins, exerting a holdase-type chaperonin function. Finally, instances of LEAP–client protein interactions have been documented, where the LEAP modulates (interferes with) the function of the client protein, acting as a surreptitious rheostat of cellular homeostasis. From the examples identified to date, it is apparent that client protein modulation also serves to mitigate stress. While some LEAPs can physically bind and protect client proteins, some apparently bind to assist the degradation of the client proteins with which they associate. Documented instances of LEAP–client protein binding, even in the absence of stress, brings to the fore the necessity of identifying how the LEAPs are degraded post-stress to render them innocuous, a first step in understanding how the cell regulates their abundance. Thermus aquaticus; [The transition from aquatic to terrestrial life overcame significant obstacles, one of which was survival in drier surroundings. This capacity, taken to its extreme, gave rise to desiccation tolerance. Constitutive “extremophile” organisms have adapted to withstand desiccation during any part of their life cycle . For most anhydrobiotes , the capacity to withstand the stresses involved in desiccation is closely associated with a single stage of their life cycle, and their cells (and thus the organism comprised of them) subsequently lose the capacity to survive this severe stress. The majority of organisms succumb to desiccation, regardless of the stage of their life cycle at which this stress occurs. The attributes that can be ascribed to the capacity to withstand desiccation have been extensively examined . The difuaticus; ). Howeveuaticus; . Specifiuaticus; . This hauaticus; ) is impluaticus; ,7,8. Eveuaticus; ,10 or deuaticus; ,12.The cellular constituents comprising the entire protective program can vary among species . The celWhat are the components of the entire protective program deployed to permit cells to survive desiccation? This list is extensive and, as previously mentioned, varies among organisms , includiProblems exist when trying to analyze the functional mechanism, at the molecular level, of the non-reducing sugars and/or LEAPs. Foremost among these is that the non-reducing sugars/LEAPs are presumed to exert their protective function in systems where water is scarce, precluding assays in hydrated environments. This has led to numerous examples of chemically altering the solution properties in which these components of the entire protective program are situated to mimic dehydration , crowdinReferring back to the previously stated premise that deployment of the entire protective program should impart desiccation tolerance to cells/organisms without this capacity, it is thought provoking to understand that there is no single entire protective program used to impart desiccation tolerance among anhydrobiotic organisms but that the components of the entire protective program can vary depending on the species! What does this imply for those hoping to impart desiccation tolerance to organisms/systems without this capacity? Are there several entire protective programs, any of which will be equally successful in providing desiccation tolerance once deployed? Conversely, does this mean that an entire protective program from one set of organisms will not work in a sub-set of different organisms, requiring the successful elucidation and deployment of a specific entire protective program tailored to a class of organisms? While the former case will assist a more rapid understanding of the fundamental requirements for desiccation tolerance that are met by multiple protective molecules, the latter eventuality will make the goal of identifying and deploying an entire protective program more daunting still!Focusing on organisms that deploy LEAPs during times of stress, there are at least two lines of thought that have evolved regarding how the LEAPs function to safeguard a stress-susceptible proteome. The first posits that they are shield molecules, physically separating cellular entities from each other during desiccation, as crowding within the dehydrating cell becomes more intense or as the opportunity of partially denatured protein aggregate formation increases . This acAlthough water seems like a simple molecule, there is still fundamental controversy regarding how this liquid sustains life . The proIf the LEAPs are forming liquid-liquid phase separation, how are client proteins recruited into these structures and is this a selective process? It is tempting to speculate that if LEAPs are essential to liquid-liquid phase separation, then each of the LEAPs involved in liquid-liquid phase separation are identifying and binding to their specific client proteins and this drives recruitment. However, this would necessitate protein–protein interaction. This would complicate the assignment of the actual protective property exerted by the LEAP in a liquid-liquid phase separation. Is the protection strictly a shield molecule functionality, housing the susceptible proteins in a liquid micro-environment, conducive to the client protein’s stability and binding only to recruit client proteins into the protective shell, or does binding to the client protein also assist in protection? Is it possible that if LEAPs set up a liquid-liquid phase separation, susceptible client proteins diffuse toward and into the more protective shell without ever engaging the LEAPs physically, constituting a legitimate shield molecule function? The addition of other solutes, e.g., specific ratios of sucrose to raffinose family oligosaccharides, may influence the propensity, timing, and types of liquid-liquid phase separation that occur as all molecular concentrations are increasing and mobility of molecules are changing as the glassy state forms during desiccation. Likewise, micro-environment alterations, and presumably liquid-liquid phase separation properties, will vary as, for example, the orthodox seed intracellular environment constantly changes through various cycles of imbibition and drying. Furthermore, as temperature alterations, and other abiotic factors, change during germination, this will also influence liquid-liquid phase separation and the protection they afford ,58.A particularly interesting characteristic of purified LEAPs is their ability to change their conformation in response to an alteration in the osmotic potential of the environment . Stress-Arabidopsis thaliana genome [Arabidopsis plants overexpressing Arabidopsis LEAPs have been tested on high NaCl. Three instances resulted in plants with superior salt tolerance at some stage of the life cycle while two instances showed reduced tolerance to NaCl (Arabidopsis LEAP OE plants were tested for NaCl tolerance (5 out of 51) is a knowledge gap due to either a lack of testing (there are 15 of the Arabidopsis LEAPs that have been overexpressed in Arabidopsis) or a lack of reporting negative results (LEAP-OEs were not more resistant to NaCl than the empty vector controls). For non-Arabidopsis LEAPs ectopically expressed in other organisms, including insect cell lines [Although accounts vary depending on how membership in the LEAP family is classified, there are at least 51 LEAPs widely acknowledged to be encoded in the a genome ,64. Ther to NaCl . The two to NaCl . The lacll lines , when tell lines . The N-tArabidopsis, there are at least seven to nine distinct families of LEAPs [The evidence supporting at least some of the LEAPs physically interacting with proteins, ostensibly to protect them from denaturation, is accumulating and is incontrovertible. LEAP–client protein physical binding offers a solution to the fact that a number of very different agronomic traits have been tightly linked to quantitative trait loci centered on genes encoding specific LEAPS . Assumiof LEAPs ,67,68 anof LEAPs ,61,69 an2O2 in this case) increases [Despite early arguments against a physical interaction between LEAPs and client proteins based on the failures of protein pulldown assays to demonstrate physical associations, subsequently, a variety of protein–protein interaction assays, with or without an applied stress, have demonstrated LEAP–client protein physical associations. These assays include yeast two hybrid ,71, phagncreases . LEAP–clThe benefit of LEAP–client protein binding has largely been assumed to be the protection of the client proteins from some form of stress; however, for reasons provided above , it is difficult to determine how LEAP binding imparts protection to a specific client protein. This quandary is very similar to that for microprotein research, where the consequences of microprotein presence are most readily explored by determining to what the microprotein binds . The samThere are instances where it seems that specific LEAPs impart protection to client proteins, binding to and preserving the client protein from permanent loss of function as and while it is stressed, and disengaging to become innocuous upon rehydration. However, other situations have been documented, where LEAP–client protein binding improves homeostasis through means other than client protein protection. So focused is the community studying LEAP function on the protective role of these intrinsically disordered proteins, we may be blind to additional consequences of LEAP–client protein binding. Is this focus on LEAP–client protein binding necessarily revealing a single LEAP functionality, that of client protein protection? Or is it diverting attention away from auxiliary functions of LEAP–client protein interaction, e.g., the modulation of cellular homeostasis? In fact, have some LEAPs capable of binding client proteins been secunded, in certain instances, to specific protein–protein interactions designed to alter client protein function to mitigate stress in addition to (or instead of) protecting the client protein from stress? Certainly, the fact that LEAPs may encompass multi-functionality has been recognized previously . Recognileap organism under stress would mimic the phenotype of the client protein in the same organism under stress, a so-called “parity of phenotypes” , interacts with the Arabidopsis Phi9 GLUTATHIONE-S-TRANSFERASE9 (At2g30860). Phi9 associates with the dehydrin in ratios of 1:5, (or greater) respectively to acquire protection from H2O2-related structural damage, presumably to the carboxy-terminus substrate binding site, which is otherwise rendered hyper-flexible by oxidation [A dehydrin from RD14 At1g180, inteRD14 At1g180, inteThe phosphorylation, or any post-translational modification (PTM) of a LEAP, demonstrates at least transient LEAP binding by a protein-modifying enzyme. However, this is quite different from the LEAP binding to impart protection or to interfere with client protein action. The LEAP-modifying enzyme, like a protease degrading a LEAP, cannot be considered a client protein based strictly on binding . NeverthArabidopsis thaliana HIRD11 (At1g54410) interacts with the Arabidopsis leucine-rich repeat receptor-like kinase (LRR-RLK) PHLOEM INTERCALATED WITH XYLEM-LIKE 1 . Plasma membrane-localized AtPXL1 phosphorylates cytoplasmically or plasma membrane-localized HIRD11. The assertion that phosphorylation leads to plasma membrane association of HIRD11 is equivocal  positively influences plant tolerance to both hypersaline (NaCl) and alkaline (carbonate) conditions when overexpressed ,81. The The SUCROSE NONFERMENTING RECEPTOR-LIKE KINASE2.10 (SnRK2.10), an SnRK2 unresponsive to ABA, binds and phosphorylates both ERD10 and ERD14 LEAPs during osmotic stress. While ERD10 maintains a cytoplasmic distribution whether it is phosphorylated or not, the population of ERD14 subsequently resides in both the cytoplasm and nucleus . The dirOryza sativa LEA 2 family gene, OsLEA5 (LOC_Os05g50710), is upregulated by ABA, at least partially due to the action of the rice ZINC FINGER C2H2 PROTEIN  in a positive feedback loop [Oryza sativa ASCORBATE PEROXIDASE 1  were found to form homo- and hetero-dimers within and among themselves, although the functional significance of these various associations has yet to be determined [How the influence of the dehydrins is exerted to maintain lipid association with the hydrophobic trans-membrane portions of PIP2B or by somehow locking the PIP2B into a stable structure in the membrane remains to be elucidated. Regardless, the overexpression of the dehydrins or their client protein aquaporin ,93 enhantermined   LEAP, GmPM28 , the closest G172800; ), somehoeratures . The SMP0, the cleratures , and interatures . CASC3 heratures , implyineratures ). The imbidopsis  while anbidopsis , and so COR15A and COR15B were knocked down by RNA interference (RNAi), a reduction in freezing tolerance, regardless of acclimation, resulted [Using immunoprecipitation to compare proteins binding to a chloroplast stroma-localized LEAP from cold-acclimated and naïve plants,  found thresulted . When RUresulted ! Equallyresulted .At least two dehydrin proteins  were found to interact with actin. ERD10 was investigated further and found to stabilize actin polymers while inhibiting both the initiation of actin growth and its depolymerization (Arabidopsis ERD10 (At1g20450) and ERD14 (At1g76180) dehydrins. MtCAS31 is upregulated by drought stress and, when CAS31 is present, it binds one of several leghemoglobin proteins  and protects MtLb120-1 against denaturation during supra-optimal temperatures and drought stress. The continued functioning of leghemoglobin during stress, safeguarded by MtCAS31, maintains the appropriate oxygen level in nodules conducive to both nitrogenase activity and bacterial respiration, supporting nodule persistence allowing the legume to retain functional nodules and nitrogen fixation capacities under drought conditions [mtcas31 suggested the protection of leghemoglobin by the dehydrin  was found to also accumulate during other abiotic stresses, including dehydration stress . The clinditions . Here, tehydrin   [MtCAS31 was overexpressed in Arabidopsis, the association of MtCAS31 with SCRM1 downregulated stomatal numbers in developing leaves, presumably by sequestering SCRM1 away from its association with other basic HELIX-LOOP-HELIX transcription factors. Because the basic HELIX-LOOP-HELIX heterodimers were not formed, the complex could not bind their cognate promoters and cell fate was altered away from the meristemoid lineage [SCRM1 overexpression results in phenotypes opposite of those for MtCAS31 overexpression (When tested in yeast two hybrid assays against an  (SCRM1) ,111) and lineage . The con lineage  . The LEAP also binds to MtAUTOPHAGY 8F  through two distinct ATG8-interacting motifs located near the amino terminus of the dehydrin. During drought stress, MtCAS31 binds the aquaporin and introduces it as cargo to the autophagosome through its simultaneous interaction with MtATG8F. Both the dehydrin and the aquaporin are degraded in this manner, reducing the water channel abundance in the plasma membrane of the roots such that hydrolytic conductivity between the plant and the soil is reduced ; Figure . Apparenatpp2-b11 RNAi lines and LEA1-overexpressing lines have enhanced ABA sensitivity. This similarity in ABA sensitivity can be explained by the observation that the AtPP2-B11 F-BOX protein targets the SUCROSE NON-FERMENTING KINASEs , reducing the titer of these kinases in the cell. These SnRKs are positive regulators of ABA signal transduction, so when they are present, ABA sensitivity is increased. For obvious reasons, reducing F-BOX titer through RNAi stabilizes the kinase titer and results in increased ABA sensitivity (Arabidopsis SKP-LIKE (ASK) proteins in E3 ligase complexes during drought stress but that the LEAP binds the FBOX to protect it during salt stress, permitting the FBOX to target a different group of proteins that would otherwise decrease salt tolerance. This conclusion requires that the FBOX have multiple target proteins (or the same target protein) that can increase (drought) or decrease  tolerance ), under normal conditions, the first generation usually had significantly reduced fertility and grain yield, relative to Wild Type. Once specific lines were chosen containing a single transgene insertion and made homozygous, the yield penalties under normal growth were eliminated while yield under drought conditions remained superior to Wild Type [The vast majority of the LEAP overexpression studies have reported enhanced fitness during stress of the organisms expressing them . For thoild Type .Arabidopsis LEAPs COR15A and COR15B, when overexpressed, both delayed flowering and resulted in a smaller rosette size relative to Wild Type [ASR1 gene in transgenic potato, tuber numbers were significantly reduced [RAB28 using a UBIQUITIN promoter. Single insertions for these lines were established from segregation ratios in back crosses to Wild Type [With this caveat in mind, the ild Type . While fild Type , the phoild Type . Althougild Type , so we a reduced . No info reduced , althougild Type . A reporild Type . In totaBy tracking LEAP persistence following the completion of germination or the alleviation of stress in normal tissues, these proteins are clearly decimated by the cells after the completion of germination ,134, upoLEAP transcript abundance, following the completion of germination, is rapidly reduced [LEAP genes are under epigenetic control, transcription from them being rapidly shut down, with rare exceptions [LEAPs are epigenetically silenced in the seed to seedling transition, some LEAPs are still available for transcription throughout the vegetative stages and so are not controlled epigenetically and remain available to assist in vegetative stress tolerance. Maintaining LEAPs available for transcription throughout the life cycle rather than epigenetically silencing them characterizes differences between the constitutive and conditional extremophiles mentioned in the introduction.One means to rapidly draw down the LEAP concentration when the proteins are no longer required is to prevent transcription and destroy the mRNA encoding them, at least ensuring that no further augmentation of the LEAP reserve is possible and assuming that the LEAPs have a high turnover rate. The  reduced , althoug reduced , and the reduced . There aceptions , followiceptions , orchestceptions ,146,147.LEAP transcription might be coupled with an active mechanism to degrade stored mRNA following imbibition. There is a report of a microRNA in tea (Camellia sinensis) targeting a LEAP transcript [LEAP of Arabidopsis [LEAP mRNA does not appear to be a general phenomenon. Other than these two examples, the assignment of microRNAs to their targets has not identified a single LEAP transcript target [Arabidopsis LEAPs is bound by RNA binding proteins, with consequences for transcript accumulation in the plant [Repressing anscript  and anotbidopsis , but miRt target  nor event target ,152. Thehe plant ,154, butEvidence that the LEAP proteins persist longer than the mRNA encoding them exists ,155, butSaccharomyces cerevisiae) were found to contain a (PEST) domain [Arabidopsis LEAP assemblage revealed few . Intriguingly, though only computer predictions, 7 of the 12 potential PESTS were present in 4 out of 10 Arabidopsis dehydrins and these four were all members of the acidic dehydrins while none of the four neutral/basic dehydrins have predicted PEST domains  domain . The PES) domain ,159. Whi) domain , a surve domains . An examectively . These hArabidopsis LEAPs are known to be differentially phosphorylated  Em protein. A cysteine endopeptidase  cuts th belong; ,167. Non belong; ). The meThe LEAPs have been determined to afford protection to cellular constituents during stressful situations. This review has focused on how LEAPs may protect a stress-susceptible proteome. Hypotheses of how this protection is provided to client proteins include a shield molecule functionality that may or may not prove to be associated with a presumed capacity for LEAPs to orchestrate liquid-liquid phase separation within the stressed cell. The shield molecule functionality does not require LEAP:client protein physical association, although recruitment of client proteins into liquid-liquid phase separation, if indeed orchestrated by LEAPs, may prove to require transient LEAP–client protein associations. Additional means by which LEAPs protect proteins include physically binding to the client protein, a chaperonin functionality that does not require the expenditure of energy, similar to the “holdases” of the heat shock protein family. Examples of such physical association leading to protection have been provided and discussed as have examples where LEAP–client protein affinity increases as the applied stress increases. This requirement of a stress-driven increase in LEAP–client protein affinity seems to explain why there is no general yield penalty for LEAP overexpression. Finally, LEAP–client protein associations may result in the modulation of client protein operation, which has unveiled a third mechanism by which LEAPs tweak the intracellular environment to maintain homeostasis in the face of stress. The identification of the client protein repertoire of individual LEAPs is imperative if the subtleties of the full range of the protective mechanism embodied in these “enigmatic” proteins is to be appreciated. Moreover, identifying the client protein repertoire of a LEAP using methods that inherently permit the identification of where in each client protein a LEAP is binding allows the client protein regions to be scrutinized for commonalities that may inform the molecular topology of the final interaction, allowing more resilient client proteins to be engineered . There i


Philadelphia-negative myeloproliferative neoplasms (MPNs) are an excellent tractable disease model of a number of aspects of human cancer biology, including genetic evolution, tissue-associated fibrosis, and cancer stem cells. In this review, we discuss recent insights into MPN biology gained from the application of a number of new single-cell technologies to study human disease, with a specific focus on single-cell genomics, single-cell transcriptomics, and digital pathology. Subsequently other mutations causing aberrant JAK-STAT signaling in MPN have been reported to occur in patients with MPN who are negative for the JAK2V617F mutation,,,JAK2V617F mutation was first described,JAK2V617F selectively promoted the growth of single-cell–derived erythropoietin-independent colonies in patients with polycythemia vera (PV).JAK2V617F mutation and a cytogenetic abnormality, demonstrating that these genetic events were present in the same clone in some patients but in separate clones in others.JAK2V617F zygosity and loss of heterozygosity breakpoints using microsatellite markers in 6495 colonies revealed that JAK2V617F homozygous clones are recurrently acquired in patients with PV and patients with essential thrombocythemia (ET).JAK2V617F first, followed by TET2 mutation, was more likely to result in a PV phenotype, typically in younger patients, but if JAK2V617F was acquired on a TET2-mutated background (“TET2-first”), an ET phenotype was more frequent. Similar observations also applied to DNMT3A and JAK2 mutations.MPN has proven to be an important disease model providing an illustration of how single-cell genomics techniques can be applied to provide new insights into disease biology and how the technology might be moved toward application for precision medicine. Indeed, to a degree, single-cell genomics approaches are already in routine clinical use in myeloid diseases through application of cytogenetic analysis, including fluorescent in situ hybridization. The most common driver mutation in MPN occurs in exon 14 of the Janus kinase 2  is the most widely applied assay in single-cell genomics, and is extensively used to provide a comprehensive and unbiased assessment of normal cellular and molecular tissue architecture and their perturbations in disease states. Over the past decade, experiments have massively expanded in their scale and implementation, due to technological advances resulting in high-throughput methods that are relatively easy to implement.,A typical analytical pipeline includes organizing cells according to their transcriptional profiles into discrete groups, or “clusters” that correlate with cell type or state.scRNAseq techniques have been widely used to study MPN, as these diseases provide an exemplar model of a cancer involving complex interactions among malignant cells, diverse immune cell types , and mesenchymal stromal cells. In chronic myeloid leukemia, stem cells were studied from patients before and after tyrosine kinase inhibitor treatment and from the same patient before and after transformation from chronic phase to blast crisis.,scRNAseq has also been applied to study how mutations alter hematopoiesis in Philadelphia-negative MPN, both in mouse models and in primary patient samples. In studies of primary cells isolated from patients with MPN, high-throughput scRNAseq has been applied to study how hematopoiesis is altered in patients with MPN.A recent study of a mouse model of mutant calreticulin (CALR)-driven ET reported similar findings. In this model, mutCALR resulted in an expansion of both HSCs and megakaryocyte progenitors, and the investigators identified an aberrant intermediary population termed “proliferative megakaryocyte progenitors (pMKP)” that fell on a distinct differentiation pathway to MKP in normal hematopoiesis in their analyses.JAK2V617F and mutCALR-driven disease also demonstrated megakaryocyte-biased hematopoiesis, with an 11-fold increase in MKP detected in patients with myelofibrosis as compared with controls in all clinical and molecular subgroups.Profiling of more than 120,000 individual cells from a range of patients with primary and secondary myelofibrosis and both Single-cell analyses have also shed light on the changes to the nonhematopoietic stromal cell compartment in MPN. Creating a “map” of certain stromal cell populations in myelofibrosis mouse models highlighted mesenchymal progenitor cells as showing the strongest upregulation in expression of extracellular matrix proteins in fibrosis. This study highlighted the S100A8/S100A9 alarmin complex as a potential therapy target, demonstrating that its inhibition was able to ameliorate the disease phenotype in the mouse model.These insights highlight the power of single-cell “omic” techniques to accurately dissect cellular and molecular perturbations in MPN. Combined with emerging techniques to capture cell states and transcriptomes in unperturbed tissues, so-called “in situ sequencing” see  single-c,The bone marrow represents a complex, dynamic, and highly regulated tissue in which diverse cell populations lie in close proximity to an orchestrated network of extracellular  matrix, blood vasculature, and bone.,,,Key morphologic features relating to marrow cellularity, megakaryocyte pleomorphism/atypia, and fibrosis are firmly embedded in current MPN classification schemes.,Computational analysis of digitized images prepared from glass slide material has evolved over the past few decades and significantly accelerated in recent years with the development of sophisticated deep learning (DL) methods that emulate the structure and function of human neurons in the form of artificial neural networks.Despite the central role of bone marrow biopsy assessment in the diagnosis and classification of MPNs, and the importance of the marrow microenvironment in disease biology (as outlined previously), few studies have attempted to apply advanced machine learning approaches to these disorders. In response, we recently demonstrated the utility of an automated image analysis pipeline that uses machine learning techniques to extract important cytomorphological and topographic features of individual megakaryocytes from digitized images of bone marrow biopsies.An important consideration in the development of improved descriptions of tissue morphology and the marrow microenvironment using digital pathology and machine learning is the development of intuitive yet sufficiently detailed data visualization. The methods used will depend on the application and requirements of the end user, but it seems likely that for clinicians and researchers unfamiliar with the normal bone marrow tissue architecture and its heterogeneity, visual representation of the cellular target(s) of interest in the context of normal and/or previously analyzed disease tissue will aid interpretation and understanding. Successfully designed outputs should capture temporal changes in the course of disease or following treatment, but also have the potential to highlight relevant diagnostic and prognostic features with attention drawn to potential therapeutic targets.Notwithstanding the potential clinical application of automated analysis of discrete single-cell populations, such as megakaryocytes in biopsies of MPN, a deeper understanding of the complex cellular interactions within the bone marrow of patients with myeloid malignancies requires a more comprehensive description of the spatiotemporal relationships that exist between the cellular and stromal components of the marrow. This will require integration of discrete cellular and extracellular morphologic features with lineage specific markers of differentiation and maturation using multiplexed immunolabeling approaches. Although several analytical platforms already use such approaches in the research setting, they are typically restricted to relatively small tissue fields  from limited sample numbers.,,Although automated analytical pipelines using convolutional neural networks to detect and segment targets of interest in digital images offer the potential for rapid sample analysis, their generation is typically dependent on access to large numbers of tissue samples accompanied by detailed clinical, laboratory, and genomic data. In general, machine learning applications in pathology use supervised approaches in which functions are learned by mapping annotated tissue features into some qualitative or quantitative output.Given the importance of accessing sufficient quantities of high-quality training and validation material, important practical considerations surround access to suitable tissue. Few centers can rely solely on locally retained tissue archives to build and validate models of discrete MPN subtypes that are sufficiently enriched with relatively rare samples corresponding to important clinical or genomic events, such as disease transformation or response to novel therapeutics. Access to trial sample cohorts and sharing of tissue libraries between collaborating clinical centers will likely optimize use of available diagnostic material and accelerate the development and validation of machine learning models of MPN.In summary, machine learning approaches to image analysis have already received broad acceptance in several branches of solid tissue pathology and are widely accepted as a transformational technology with significant clinical and research potential. Realizing this potential in MPN will depend on the identification and extraction of important cell-cell and cell-stroma interactions that complement and enhance our understanding of the dynamics underlying the clonal expansion of single-cell precursors that ultimately drive the disease phenotype in individual patients. This will require close collaboration among hematologists, pathologists, bioinformaticians, biomedical engineers, and software engineers and the integration of multimodality approaches spanning novel single-cell and whole-tissue sample technologies.JAK2V617F show such heterogeneity in fitness advantage. This is crucial to understand if we are to develop treatment approaches that reverse the fitness advantage to induce molecular responses and ultimately alter the natural history of MPN. Another key challenge in the field is to translate these single-cell technological developments through to direct patient benefit. Although at the present time it may seem farfetched for single-cell methodologies to be applied routinely in clinical diagnostics, this will surely become a reality over the coming years with obvious utility for improved approaches to diagnose and classify disease as well as monitor response to treatment and predict risk of disease progression.Single-cell technologies have over many years provided remarkable insights into MPN biology, making conceptual advances of broader relevance across cancer biology. Future technological developments in digital pathology, in situ sequencing, and single-cell multiomics approaches see  will undNone.