Structure of the thrombopoietin-MPL receptor complex is a blueprint for biasing hematopoiesis.

Thrombopoietin (THPO or TPO) is an essential cytokine for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Here, we report the 3.4 Å resolution cryoelectron microscopy structure of the extracellular TPO-TPO receptor (TpoR or MPL) signaling complex, revealing the basis for homodimeric MPL activation and providing a structural rationalization for genetic loss-of-function thrombocytopenia mutations. The structure guided the engineering of TPO variants (TPOmod) with a spectrum of signaling activities, from neutral antagonists to partial- and super-agonists. Partial agonist TPOmod decoupled JAK/STAT from ERK/AKT/CREB activation, driving a bias for megakaryopoiesis and platelet production without causing significant HSC expansion in mice and showing superior maintenance of human HSCs in vitro. These data demonstrate the functional uncoupling of the two primary roles of TPO, highlighting the potential utility of TPOmod in hematology research and clinical HSC transplantation.

Detection of Rehabilitation Training Effect of Upper Limb Movement Disorder Based on MPL-CNN.

Stroke represents a medical emergency and can lead to the development of movement disorders such as abnormal muscle tone, limited range of motion, or abnormalities in coordination and balance. In order to help stroke patients recover as soon as possible, rehabilitation training methods employ various movement modes such as ordinary movements and joint reactions to induce active reactions in the limbs and gradually restore normal functions. Rehabilitation effect evaluation can help physicians understand the rehabilitation needs of different patients, determine effective treatment methods and strategies, and improve treatment efficiency. In order to achieve real-time and accuracy of action detection, this article uses Mediapipe's action detection algorithm and proposes a model based on MPL-CNN. Mediapipe can be used to identify key point features of the patient's upper limbs and simultaneously identify key point features of the hand. In order to detect the effect of rehabilitation training for upper limb movement disorders, LSTM and CNN are combined to form a new LSTM-CNN model, which is used to identify the action features of upper limb rehabilitation training extracted by Medipipe. The MPL-CNN model can effectively identify the accuracy of rehabilitation movements during upper limb rehabilitation training for stroke patients. In order to ensure the scientific validity and unified standards of rehabilitation training movements, this article employs the postures in the Fugl-Meyer Upper Limb Rehabilitation Training Functional Assessment Form (FMA) and establishes an FMA upper limb rehabilitation data set for experimental verification. Experimental results show that in each stage of the Fugl-Meyer upper limb rehabilitation training evaluation effect detection, the MPL-CNN-based method's recognition accuracy of upper limb rehabilitation training actions reached 95%. At the same time, the average accuracy rate of various upper limb rehabilitation training actions reaches 97.54%. This shows that the model is highly robust across different action categories and proves that the MPL-CNN model is an effective and feasible solution. This method based on MPL-CNN can provide a high-precision detection method for the evaluation of rehabilitation effects of upper limb movement disorders after stroke, helping clinicians in evaluating the patient's rehabilitation progress and adjusting the rehabilitation plan based on the evaluation results. This will help improve the personalization and precision of rehabilitation treatment and promote patient recovery.

Rli51 Attenuates Transcription of the Listeria Pathogenicity Island 1 Gene mpl and Functions as a Trans-Acting sRNA in Intracellular Bacteria.

Listeria pathogenicity island 1 (LIPI-1) is a genetic region containing a cluster of genes essential for virulence of the bacterial pathogen Listeria monocytogenes. Main virulence factors in LIPI-1 include long 5' untranslated regions (5'UTRs), among which is Rli51, a small RNA (sRNA) in the 5'UTR of the Zn-metalloprotease-coding mpl. So far, Rli51 function and molecular mechanisms have remained obscure. Here, we show that Rli51 exhibits a dual mechanism of regulation, functioning as a cis- and as a trans-acting sRNA. Under nutrient-rich conditions, rli51-mpl transcription is prematurely terminated, releasing a short 121-nucleotide-long sRNA. Rli51 is predicted to function as a transcription attenuator that can fold into either a terminator or a thermodynamically more stable antiterminator. We show that the sRNA Rli21/RliI binds to a single-stranded RNA loop in Rli51, which is essential to mediate premature transcription termination, suggesting that sRNA binding could stabilize the terminator fold. During intracellular infection, rli51 transcription is increased, which generates a higher abundance of the short Rli51 sRNA and allows for transcriptional read-through into mpl. Comparative intracellular bacterial transcriptomics in rli51-null mutants and the wild-type reference strain EGD-e suggests that Rli51 upregulates iron-scavenging proteins and downregulates virulence factors from LIPI-1. MS2 affinity purification confirmed that Rli51 binds transcripts of the heme-binding protein Lmo2186 and Lmo0937 in vivo. These results prove that Rli51 functions as a trans-acting sRNA in intracellular bacteria. Our research shows a growth condition-dependent mechanism of regulation for Rli51, preventing unintended mpl transcription in extracellular bacteria and regulating genes important for virulence in intracellular bacteria.

Effects of calreticulin mutations on cell transformation and immunity.

Myeloproliferative neoplasms (MPNs) are cancers involving dysregulated production and function of myeloid lineage hematopoietic cells. Among MPNs, Essential thrombocythemia (ET), Polycythemia Vera (PV) and Myelofibrosis (MF), are driven by mutations that activate the JAK-STAT signalling pathway. Somatic mutations of calreticulin (CRT), an endoplasmic reticulum (ER)-localized lectin chaperone, are driver mutations in approximately 25% of ET and 35% of MF patients. The MPN-linked mutant CRT proteins have novel frameshifted carboxy-domain sequences and lack an ER retention motif, resulting in their secretion. Wild type CRT is a regulator of ER calcium homeostasis and plays a key role in the assembly of major histocompatibility complex (MHC) class I molecules, which are the ligands for antigen receptors of CD8+ T cells. Mutant CRT-linked oncogenesis results from the dysregulation of calcium signalling in cells and the formation of stable complexes of mutant CRT with myeloproliferative leukemia (MPL) protein, followed by downstream activation of the JAK-STAT signalling pathway. The intricate participation of CRT in ER protein folding, calcium homeostasis and immunity suggests the involvement of multiple mechanisms of mutant CRT-linked oncogenesis. In this review, we highlight recent findings related to the role of MPN-linked CRT mutations in the dysregulation of calcium homeostasis, MPL activation and immunity.

In Silico Evaluation of Coding and Non-Coding nsSNPs in the Thrombopoietin Receptor (MPL) Proto-Oncogene: Assessing Their Influence on Protein Stability, Structure, and Function.

The thrombopoietin receptor (MPL) gene is a critical regulator of hematopoiesis, and any alterations in its structure or function can result in a range of hematological disorders. Non-synonymous single nucleotide polymorphisms (nsSNPs) in MPL have the potential to disrupt normal protein function, prompting our investigation into the most deleterious MPL SNPs and the associated structural changes affecting protein-protein interactions. We employed a comprehensive suite of bioinformatics tools, including PredictSNP, InterPro, ConSurf, I-Mutant2.0, MUpro, Musitedeep, Project HOPE, STRING, RegulomeDB, Mutpred2, CScape, and CScape Somatic, to analyze 635 nsSNPs within the MPL gene. Among the analyzed nsSNPs, PredictSNP identified 28 as significantly pathogenic, revealing three critical functional domains within MPL. Ten of these nsSNPs exhibited high conservation scores, indicating potential effects on protein structure and function, while 14 were found to compromise MPL protein stability. Although the most harmful nsSNPs did not directly impact post-translational modification sites, 13 had the capacity to substantially alter the protein's physicochemical properties. Some mutations posed a risk to vital protein-protein interactions crucial for hematological functions, and three non-coding region nsSNPs displayed significant regulatory potential with potential implications for hematopoiesis. Furthermore, 13 out of 21 nsSNPs evaluated were classified as high-risk pathogenic variants by Mutpred2. Notably, amino acid alterations such as C291S, T293N, D295G, and W435C, while impactful on protein stability and function, were deemed non-oncogenic "passenger" mutations. Our study underscores the substantial impact of missense nsSNPs on MPL protein structure and function. Given MPL's central role in hematopoiesis, these mutations can significantly disrupt hematological processes, potentially leading to a variety of disorders. The identified high-risk pathogenic nsSNPs may hold promise as potential biomarkers or therapeutic targets for hematological diseases. This research lays the foundation for future investigations into the MPL gene's role in the realm of hematological health and diseases.

A novel mutation in MECOM affects MPL regulation in vitro and results in thrombocytopenia and bone marrow failure.

MECOM-associated syndrome (MECOM-AS) is a rare disease characterized by amegakaryocytic thrombocytopenia, progressive bone marrow failure, pancytopenia and radioulnar synostosis with high penetrance. The clinical phenotype may also include finger malformations, cardiac and renal alterations, hearing loss, B-cell deficiency and predisposition to infections. The syndrome, usually diagnosed in the neonatal period because of severe thrombocytopenia, is caused by mutations in the MECOM gene, encoding for the transcription factor EVI1. The mechanism linking the alteration of EVI1 function and thrombocytopenia is poorly understood. In a paediatric patient affected by severe thrombocytopenia, we identified a novel variant of the MECOM gene (p.P634L), whose effect was tested on pAP-1 enhancer element and promoters of targeted genes showing that the mutation impairs the repressive activity of the transcription factor. Moreover, we demonstrated that EVI1 controls the transcriptional regulation of MPL, a gene whose mutations are responsible for congenital amegakaryocytic thrombocytopenia (CAMT), potentially explaining the partial overlap between MECOM-AS and CAMT.

Interplay between MYZUS PERSICAE-INDUCED LIPASE 1 and OPDA signaling in limiting green peach aphid infestation on Arabidopsis thaliana.

MYZUS PERSICAE-INDUCED LIPASE1 (MPL1) encodes a lipase in Arabidopsis thaliana that is required for limiting infestation by the green peach aphid (GPA; Myzus persicae), an important phloem sap-consuming insect pest. Previously, we demonstrated that MPL1 expression was up-regulated in response to GPA infestation, and GPA fecundity was higher on the mpl1 mutant, compared with the wild-type (WT), and lower on 35S:MPL1 plants that constitutively expressed MPL1 from the 35S promoter. Here, we show that the MPL1 promoter is active in the phloem and expression of the MPL1 coding sequence from the phloem-specific SUC2 promoter in mpl1 is sufficient to restore resistance to GPA. The GPA infestation-associated up-regulation of MPL1 requires CYCLOPHILIN 20-3 (CYP20-3), which encodes a 12-oxo-phytodienoic acid (OPDA)-binding protein that is involved in OPDA signaling, and is required for limiting GPA infestation. OPDA promotes MPL1 expression to limit GPA fecundity, a process that requires CYP20-3 function. These results along with our observation that constitutive expression of MPL1 from the 35S promoter restores resistance to GPA in the cyp20-3 mutant, and MPL1 acts in a feedback loop to limit OPDA levels in GPA-infested plants, suggest that an interplay between MPL1, OPDA, and CYP20-3 contributes to resistance to GPA.

Genomic and computational analysis of four novel variants of MPL gene in Congenital Amegakaryocytic Thrombocytopenia.

Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare, genetic, autosomal recessive disorder characterized by severe thrombocytopenia, due to inefficient bone marrow megakaryopoiesis eventually leading to aplasia. Majority of the cases are due to homozygous or compound heterozygous mutations in MPL gene encoding for thrombopoietin (THPO) receptor protein. CAMT can be diagnosed at early phase of life, with major complication of transfusion dependency and hematopoietic transplantation as only curative treatment. We have investigated the sequence variations in MPL gene of 7 bone marrow failure (BMF) subjects, who presented with clinically diverse phenotypes, through next generation sequencing (NGS). Plasma THPO levels were estimated using ELISA. Insilico sequence and structure-based analyses were performed to understand the structural and functional implications of mutations, identified through NGS. We studied 7 CAMT subjects suspected of BMF, who presented with severe thrombocytopenia followed by pancytopenia, bleeding manifestation and physical anomalies. The plasma THPO levels were significantly elevated (p<0.05) in all the cases. Molecular analysis by NGS identified 9 genomic mutations in MPL gene. These included 7 non-synonymous substitution, 1 nonsense substitution and 1 in-del mutations, of which 4 are novel mutations. Insilico analysis predicted damaging effects on THPO-R and its reduced affinity for THPO for all the identified mutations. CAMT is a rare disorder with diverse clinical phenotypes and diagnosis is challenging. The elevated plasma THPO levels should be considered for the primary diagnosis and prognosis of the disease. However, molecular analysis of MPL gene is important for the diagnosis and management of the disease through genetic counselling. Though the cytokines, THPO-R agonist are used for the treatment of CAMT, HSCT is the only curative therapy.

Intranasal Monophosphoryl Lipid a Administration Ameliorates depression-like Behavior in Chronically Stressed Mice Through Stimulation of Microglia.

We and others have reported that systematic stimulation of the central innate immune system by a low dose of lipopolysaccharide (LPS) can improve depression-like behavior in chronically stressed animals. However, it is unclear whether similar stimulation by intranasal administration could improve depression-like behavior in animals. We investigated this question using monophosphoryl lipid A (MPL), a derivative of LPS that lacks the adverse effects of LPS but is still immuno-stimulatory. We found that a single intranasal administration of MPL at a dose of 10 or 20 µg/mouse, but not at a dose of 5 µg/mouse, ameliorated chronic unpredictable stress (CUS)-induced depression-like behavior in mice, as evidenced by the decrease in immobility time in tail suspension test and forced swimming test and the increase in sucrose intake in sucrose preference test. In the time-dependent analysis, the antidepressant-like effect of a single intranasal MPL administration (20 µg/mouse) was observed 5 and 8 h but not 3 h after drug administration and persisted for at least 7 days. Fourteen days after the first intranasal MPL administration, a second intranasal MPL administration (20 µg/mouse) still showed an antidepressant-like effect. The innate immune response mediated by microglia might mediate the antidepressant-like effect of intranasal MPL administration, because both inhibition of microglial activation by pretreatment with minocycline and depletion of microglia by pretreatment with PLX3397 prevented the antidepressant-like effect of intranasal MPL administration. These results suggest that intranasal administration of MPL can produce significant antidepressant-like effects in animals under chronic stress conditions via stimulation of microglia.

Metabolic engineering of Escherichia coli to efficiently produce monophosphoryl lipid A.

Monophosphoryl lipid A (MPL), mainly isolated from Salmonella minnesota R595, has been used as adjuvant in several vaccines. In this study, an Escherichia coli strain that can efficiently produce the MPL has been constructed. The gene clusters related to the biosynthesis of O-antigen, core oligosaccharide, enterobacterial common antigen, and colanic acid were sequentially removed to save the carbon source and to increase the activity of PagP in E. coli MG1655. Then, the genes pldA, mlaA, and mlaC related to the phospholipid transport system were further deleted, resulting in the strain MW012. Finally, the genes lpxE from Francisella novicida and pagP and pagL from Salmonella were overexpressed in MW012 to modify the structure of lipid A, resulting in the strain MW012/pWEPL. Lipid A species were isolated from MW012/pWEPL and analyzed by thin-layer chromatography and liquid chromatography-mass spectrometry. The results showed that mainly two MPL species were produced in E. coli MW012/pWEPL, one is hexa-acylated, and the other is penta-acylated. More importantly, the proportion of the hexa-acylated MPL, which is the most effective component of lipid A vaccine adjuvant, reached 75%. E. coli MW012/pWEPL constructed in this study provided a good alternative for the production of lipid A vaccine adjuvant MPL.

Genomics of clonal evolution in a rare essential thrombocythemia with coexisting Type 2 CALR and MPL S204P mutations.

Essential thrombocythemia (ET) with double driver mutations is a rare disease. ET patients with both MPL and Type 1 CALR mutations have been reported. Here, we report the first case of an ET patient with both MPL S204P and Type 2 CALR mutations and a summary of our literature review findings. In the patient whose case is reported here, the disease progressed to an accelerated phase 3.5 months after diagnosis. CALR mutation disappeared and new mutations emerged as the disease progressed, such as ASXL1, CBL, ETV6, and PTPN11 mutations. This case highlights that screening for additional mutations using NGS should be considered in patients with ET to assess the prognosis, especially as the disease progresses.

Frequency of JAK2V617F, MPL and CALR driver mutations and associated clinical characteristics in a Norwegian patient cohort with myeloproliferative neoplasms.

Myeloproliferative neoplasms are hematological disorders characterized by increased production in one or more myeloid cell lines, associated with driver mutations in JAK2-, MPL- and CALR-genes. The aims of this study were to investigate the prevalence of these driver mutations in a Norwegian patient cohort with myeloproliferative neoplasms, and to assess whether the different mutations were associated with different clinical presentation and natural history.Results from 820 patients in whom analysis for JAK2V617F-, CALR- and MPL had been performed at Haukeland University Hospital in the period 2014-2019 were retrieved and analyzed together with clinical variables related to diagnosis, hematological blood parameters and complications, obtained from patient records.We identified 182 cases of myeloproliferative neoplasms: 78 with JAK2V617F, 28 with CALR-mutations, two with MPL-mutations and 23 cases without a driver mutation. There was a lower prevalence of JAK2V617F mutation than expected in the polycythemia vera group, likely related to overdiagnosis. In patients with essential thrombocytosis, we found significantly higher levels of hemoglobin and erythrocyte volume fraction for JAK2V617F-mutated disease, and significantly higher levels of platelets and lactate dehydrogenase for CALR-mutated disease. Patients with JAK2V617F-mutated primary myelofibrosis had significantly higher levels of hemoglobin, and there was an increased number of smokers or former smokers in this group compared to patients with CALR-mutations.Except for a lower prevalence of JAK2V617F-mutation in polycythemia vera, the mutational distribution in our patient cohort was similar to previous findings in other populations. The novel finding of a higher prevalence of smokers in JAK2V617F-mutated primary myelofibrosis warrants further investigation.

Advances in molecular evaluation of myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPN) are a group of clonal hematopoietic stem cell disorders with uncontrolled proliferation of one or more hematopoietic cell types, including myeloid, erythroid and megakaryocytic lineages, and minimal defect in maturation. Most MPN are associated with well-defined molecular abnormalities involving genes that encode protein tyrosine kinases that lead to constitutive activation of the downstream signal transduction pathways and confer cells proliferative and survival advantage. Genome-wide sequencing analyses have discovered secondary cooperating mutations that are shared by most of the MPN subtypes as well as other myeloid neoplasms and play a major role in disease progression. Without appropriate management, the natural history of most MPN consists of an initial chronic phase and a terminal blast phase. Molecular aberrations involving protein tyrosine kinases have been used for the diagnosis, classification, detection of minimal/measurable residual disease, and target therapy. We review recent advances in molecular genetic aberrations in MPN with a focus on MPN associated with gene rearrangements or mutations involving tyrosine kinase pathways.

Expanded molecular detection of MPL codon p.W515 and p.S505N mutations in myeloproliferative neoplasms.

BACKGROUND: Patients negative for the JAK2 p.V617F somatic variant are frequently reflexed to testing for MPL exon 10 variants. Detection of these variants via multiplexed allele-specific PCR followed by fragment analysis has been previously published. The present study builds on this concept by improving the detection of the p.W515A variant, adding a second allele-specific primer to detect the p.W515R variant, and incorporating an improved primer for p.S505N detection. METHODS: The W515 amplification employs 5'-labeled allele-specific forward primers to detect p.W515K, p.W515L, p.W515R, and p.W515A. The p.S505N amplification includes an allele-specific reverse primer with a tail extension. Fragments were subject to capillary electrophoresis on an ABI 3500 Genetic Analyzer and analyzed using GeneMapper 6.0 (Thermo Fisher Scientific). RESULTS: Thirty MPL-negative and 13 MPL-positive samples previously tested by a reference laboratory were tested with the MPL LDT. Results were 100% concordant. The MPL LDT has a limit of detection of at least 5% VAF for the p.W515 variants and 10% VAF for the p.S505N variant. CONCLUSION: Current MPL assays are predominantly focused on p.W515L/K and p.S505N mutations. We have engineered an MPL test for detecting p.W515A/L/K/R and p.S505N variants, thereby increasing the diagnostic yield with little additional expense or technician time.

Comprehensive genomic analysis of the potential limitations of several published PCR primers targeting prfA-virulence gene cluster in Listeria species.

Polymerase chain reaction (PCR) is commonly used to detect Listeria monocytogenes, foodborne pathogen. This study conducted in silico genomic analysis to investigate the specificity and binding efficacy of four published pairs of PCR primers targeting Listeria prfA-virulence gene cluster (pVGC) based on Listeria sequences available. We first performed comprehensive genomic analyses of the pVGC, the main pathogenicity island in Listeria spp. In total, 2961 prfA, 642 plcB, 629 mpl, and 1181 hlyA gene sequences were retrieved from the NCBI database. Multiple sequence alignments and phylogenetic trees were generated using unique (non-identical or not-shared) sequences of each represented genes, targeting four pairs of PCR primers published previously, namely 202 prfA, 82 plcB, 150 mpl, and 176 hlyA unique gene sequences. Only the hlyA gene showed strong (over 94%) primer mapping results, while prfA, plcB, and mpl genes showed weak (<50%) matching results. In addition, nucleotide variations were observed at the 3' end of the primers, indicating non-binding to the targets could potentially cause false-negative results. Thus, we propose designing degenerate primers or multiple PCR primers based on as many isolates as possible to minimize the false-negative risk and reach the aim of low tolerable limits of detection.

Pathogenic "germline" variants associated with myeloproliferative disorders in apparently normal individuals: Inherited or acquired genetic alterations?

Myeloproliferative syndromes (MPS) are hematologic malignancies due to the expansion of an abnormal hematopoietic stem cell. They include chronic myeloid leukemia (CML) and non-CML MPS such as polycythemia vera, essential thrombocythemia and primary myelofibrosis. The latter are distinguished by somatic pathogenic variants affecting JAK2, CALR, or MPL genes. Apparent germline pathogenic variants have been reported in the general population. Here, we found that two gnomAD data-sets report more homozygotes than expected for the JAK2 c.1849G > T(Val617Phe) variant. We propose that somatic gene conversion can explain the presence of those unexpected homozygotes in normal populations. Consistently, homozygous individuals are older than 65 years. We also found a lower-than-expected frequency of the JAK2 variant in younger individuals suggesting that somatic mutation can underlie its presence in (at least some) heterozygotes. Regarding pathogenic variants in MPL and CALR, they are also present in the gnomAD data-sets explored. However, we cannot conclude that such seemingly germline variants are in fact somatic alterations. These results suggest that apparently normal individuals bearing MPS-related variants can be subclinical/undiagnosed MPS cases of somatic origin. It would be interesting to assess the hematologic phenotype of such individuals and the presence of the relevant variants in other tissues.

Clonogenic assays improve determination of variant allele frequency of driver mutations in myeloproliferative neoplasms.

Molecular diagnostics moves more into focus as technology advances. In patients with myeloproliferative neoplasms (MPN), identification and monitoring of the driver mutations have become an integral part of diagnosis and monitoring of the disease. In some patients, none of the known driver mutations (JAK2V617F, CALR, MPL) is found, and they are termed "triple negative" (TN). Also, whole-blood variant allele frequency (VAF) of driver mutations may not adequately reflect the VAF in the stem cells driving the disease. We reasoned that colony forming unit (CFU) assay-derived clonogenic cells may be better suited than next-generation sequencing (NGS) of whole blood to detect driver mutations in TN patients and to provide a VAF of disease-driving cells. We have included 59 patients carrying the most common driver mutations in the establishment or our model. Interestingly, cloning efficiency correlated with whole blood VAF (p = 0.0048), suggesting that the number of disease-driving cells correlated with VAF. Furthermore, the clonogenic VAF correlated significantly with the NGS VAF (p < 0.0001). This correlation was lost in patients with an NGS VAF <15%. Further analysis showed that in patients with a VAF <15% by NGS, clonogenic VAF was higher than NGS VAF (p = 0.003), suggesting an enrichment of low numbers of disease-driving cells in CFU assays. However, our approach did not enhance the identification of driver mutations in 5 TN patients. A significant correlation of lactate dehydrogenase (LDH) serum levels with both CFU- and NGS-derived VAF was found. Our results demonstrate that enrichment for clonogenic cells can improve the detection of MPN driver mutations in patients with low VAF and that LDH levels correlate with VAF.

Augmentation of NK Cell Proliferation and Anti-tumor Immunity by Transgenic Expression of Receptors for EPO or TPO.

Many investigational adoptive immunotherapy regimens utilizing natural killer (NK) cells require the administration of interleukin-2 (IL-2) or IL-15, but these cytokines cause serious dose-dependent toxicities. To reduce or preclude the necessity for IL-2 use, we investigated whether genetic engineering of NK cells to express the erythropoietin (EPO) receptor (EPOR) or thrombopoietin (TPO) receptor (c-MPL) could be used as a method to improve NK cell survival and function. Viral transduction of NK-92 cells to express EPOR or c-MPL receptors conveyed signaling via appropriate pathways, protected cells from apoptosis, augmented cellular proliferation, and increased cell cytotoxic function in response to EPO or TPO ligands in vitro. In the presence of TPO, viral transduction of primary human NK cells to express c-MPL enhanced cellular proliferation and increased degranulation and cytokine production toward target cells in vitro. In contrast, transgenic expression of EPOR did not augment the proliferation of primary NK cells. In immunodeficient mice receiving TPO, in vivo persistence of primary human NK cells genetically modified to express c-MPL was higher compared with control NK cells. These data support the concept that genetic manipulation of NK cells to express hematopoietic growth factor receptors could be used as a strategy to augment NK cell proliferation and antitumor immunity.

Pro106Leu MPL mutation is associated with thrombocytosis and a low risk of thrombosis, splenomegaly and marrow fibrosis.

The P106L mutation in the human myeloproliferative leukemia virus oncogene (MPL) was shown to be associated with hereditary thrombocythemia in Arabs. The clinical and bone marrow (BM) features of P106L mutation are unknown. Genetic databases at two tertiary hospitals in Saudi Arabia were searched to identify patients with the MPL P106L mutation. Clinical data were collected retrospectively and the BM aspirates and biopsies were independently reviewed by two hematopathologists. In total, 115 patients were included. Median age was 33 years of which 31 patients were pediatric and 65 were female. The mutation was homozygous in 87 patients. Thrombocytosis was documented in 107 patients, with a median platelet count of 667 × 109/L. The homozygous genotype was associated with a higher platelet count. Thirty-three patients had an evaluable BM and clustering of megakaryocytes was observed in 30/33 patients. At the time of last follow-up, 114 patients were alive. The median follow-up was 7.8 years from the time of thrombocytosis. No patients developed disease progression to myelofibrosis. The P106L mutation was associated with marked thrombocytosis at a younger age and with a low risk of thrombosis, splenomegaly, and marrow fibrosis. The BM demonstrated normal or hypocellular marrow with megakaryocyte clusters.

Philadelphia-positive acute lymphoblastic leukemia in a case of MPL p.(W515L) variant essential thrombocythemia: case report and literature review.

Acute lymphoblastic leukemia (ALL) arising in preexisting myeloproliferative neoplasms (MPN) is rare with historical cases unable to differentiate between concomitant malignancies or leukemic transformation. Here, we report a case of patient with Philadelphia positive B lymphoblastic leukemia (Ph+ALL) who developed from MPL-mutated essential thrombocythemia (ET) 13 years after initial presentation. Molecular studies showed the discrepancy between the high percentage of lymphocyte blasts (91%) and the low MPL p.(W515L) variant allele frequency (2.59%) at diagnosis in the bone marrow, indicating that the Ph+ALL clone did not originate from the ET clone carrying the MPL p.(W515L) variant. After the treatment of a new tyrosine kinase inhibitor flumatinib and prednisolone, cytogenetic and molecular remission had been achieved rapidly and followed by the recovery of original ET manifestation. Although relapsed eventually, this is still a very rare case of simultaneous presence of two cytogenetics abnormalities and evolution of MPL p.(W515L) variant ET to Ph+ALL and may provide evidence to illustrate the clonal relationship of MPN and post-MPN ALL.