Abnormal expression of CUX1 influences autophagy activation in paroxysmal nocturnal hemoglobinuria.

The mechanism underlying autophagy in paroxysmal nocturnal hemoglobinuria (PNH) remains largely unknown. We previously sequenced the entire genome exon of the CD59- cells from 13 patients with PNH and found genes such as CUX1 encoding Cut-like homeobox 1. Peripheral blood samples from 9 patients with PNH and 7 healthy control subjects were obtained to measure CUX1 expression. The correlation between CUX1 messenger RNA expression and PNH clinical indicators was analyzed. To simulate CUX1 expression in patients with PNH, we generated a panel of PNH cell lines by knocking out PIGA in K562 cell lines and transfected lentivirus with CUX1. CCK-8 and EDU assay assessed cell proliferation. Western blotting was used to detect Beclin-1, LC3A, LC3B, ULK1, PI3K, AKT, p-AKT, mTOR, and p-mTOR protein levels. Autophagosomes were observed with transmission electron microscopy. Chloroquine was used to observe CUX1 expression in PNH after autophagy inhibition. Leukocytes from patients with PNH had lower levels of CUX1 messenger RNA expression and protein content than healthy control subjects. The lactose dehydrogenase level and the percentage of PNH clones were negatively correlated with CUX1 relative expression. We reduced CUX1 expression in a PIGA knockout K562 cell line, leading to increased cell proliferation. Levels of autophagy markers Beclin-1, LC3B, LC3A, and ULK1 increased, and autophagosomes increased. Furthermore, PI3K/AKT/mTOR protein phosphorylation levels were lower. CUX1 expression did not change and cell proliferation decreased in CUX1 knocked down PNH cells after inhibition of autophagy by chloroquine. In brief, CUX1 loss-of-function mutation resulted in stronger autophagy in PNH.

A Journey from Blood Cells to Genes and Back.

I was attracted to hematology because by combining clinical findings with the use of a microscope and simple laboratory tests, one could often make a diagnosis. I was attracted to genetics when I learned about inherited blood disorders, at a time when we had only hints that somatic mutations were also important. It seemed clear that if we understood not only what genetic changes caused what diseases but also the mechanisms through which those genetic changes contribute to cause disease, we could improve management. Thus, I investigated many aspects of the glucose-6-phosphate dehydrogenase system, including cloning of the gene, and in the study of paroxysmal nocturnal hemoglobinuria (PNH), I found that it is a clonal disorder; subsequently, we were able to explain how a nonmalignant clone can expand, and I was involved in the first trial of PNH treatment by complement inhibition. I was fortunate to do clinical and research hematology in five countries; in all of them, I learned from mentors, from colleagues, and from patients.

Complement Inhibitors in the Management of Complement-Mediated Hemolytic Uremic Syndrome and Paroxysmal Nocturnal Hemoglobinuria.

BACKGROUND: Complement-mediated HUS (CM-HUS) and paroxysmal nocturnal hemoglobinuria (PNH) are rare hematologic disorders that cause dysregulation and hyperactivation of the complement system. Historically, treatment of CM-HUS involved plasma exchange (PLEX), often with limited benefit and variable tolerance. Conversely, PNH was treated with supportive care or hemopoietic stem cell transplant. Within the last decade, monoclonal antibody therapies that block terminal complement pathway activation, have emerged as less invasive and more efficacious options for management of both disorders. This manuscript seeks to discuss a relevant clinical case of CM-HUS and the evolving landscape of complement inhibitor therapies for CM-HUS and PNH. AREAS OF UNCERTAINTY: Eculizumab, the first humanized anti-C5 monoclonal antibody, has been the standard of care in treating CM-HUS and PNH for over a decade. Although eculizumab has remained an effective agent, the variability in ease and frequency of administration has remained an obstacle for patients. The development of novel complement inhibitor therapies with longer half-lives, has allowed for changes in frequency and route of administration, thus improving patient QOL. However, there are limited prospective clinical trial data given disease rarity, and limited information on variable infusion frequency and length of treatment. THERAPEUTIC ADVANCES: Recently, there has been a push to formulate complement inhibitors that improve QOL while maintaining efficacy. Ravulizumab, a derivative of eculizumab, was developed to allow for less frequent administration, while remaining efficacious. In addition, the novel oral and subcutaneous therapies, danicopan and crovalimab, respectively, along with pegcetacoplan are currently undergoing active clinical trials, and poised to further reduce treatment burden. CONCLUSION: Complement inhibitor therapies have changed the treatment landscape for CM-HUS and PNH. With a significant emphasis on patient QOL, novel therapies continue to emerge and require an in-depth review of their appropriate use and efficacy in these rare disorders. CLINICAL CASE: A 47-year-old woman with hypertension and hyperlipidemia presented with shortness of breath and was found to have hypertensive emergency in the setting of acute renal failure. Her serum creatinine was 13.9 mg/dL; elevated from 1.43 mg/dL 2 years before. The differential diagnosis for her acute kidney injury (AKI) included infectious, autoimmune, and hematologic processes. Infectious work-up was negative. ADAMTS13 activity level was not low at 72.9%, ruling out thrombotic thrombocytopenic purpura (TTP). Patient underwent a renal biopsy, which revealed acute on chronic thrombotic microangiopathy (TMA). A trial of eculizumab was initiated with concurrent hemodialysis. The diagnosis of CM-HUS was later confirmed by a heterozygous mutation in complement factor I (CFI), resulting in increased membrane attack complex (MAC) cascade activation. The patient was maintained on biweekly eculizumab and was eventually transitioned to ravulizumab infusions as an outpatient. Her renal failure did not recover, and the patient remains on hemodialysis while awaiting kidney transplantation.

Fatigue and health-related quality of life in paroxysmal nocturnal haemoglobinuria: A post hoc analysis of the pegcetacoplan PEGASUS trial data.

OBJECTIVES: Paroxysmal nocturnal haemoglobinuria (PNH) is a rare, non-malignant haematological disorder associated with disabling fatigue and reduced health-related quality of life. Post hoc analysis of PEGASUS phase 3 trial (NCT03500549) characterised improvements in patient-reported fatigue measured by functional assessment of chronic illness therapy-fatigue (FACIT-fatigue) instrument item-level ratings for pegcetacoplan and eculizumab for the treatment of PNH. METHODS: Item-level responder analysis was conducted on a ≥2-level change from baseline (CFB) clinically important response (CIR) for the FACIT-fatigue 13 individual items rated on a 5-level Likert scale. We evaluated ≥2-level change against the minimal clinically important difference (MCID) of the FACIT-fatigue total score (≥5 points) and clinical parameters, haemoglobin (Hb; ≥1 g/dL) and normalised absolute reticulocyte count (ARC; 30-100 pg/cells). Logistic regressions estimated baseline-to-Week-16 FACIT-fatigue item-level transitional probabilities; Kaplan-Meier analysis estimated time to FACIT-fatigue item CIR. RESULTS: Pegcetacoplan versus eculizumab was associated with significantly greater odds of Week 16 CIR across 8/13 items and on total score MCID (OR [CI] = 11.19 [3.73, 33.57]) and faster times to responses. The item-level CIR threshold also showed clinical relevance on Hb level and ARC normalization. CONCLUSIONS: Compared with eculizumab, pegcetacoplan was associated with clinically meaningful greater improvements on a majority of FACIT-fatigue items.

Molecular landscape of immune pressure and escape in aplastic anemia.

Idiopathic aplastic anemia (IAA) pathophysiology is dominated by autoreactivity of human leukocyte antigen (HLA)-restricted T-cells against antigens presented by hematopoietic stem and progenitor cells (HSPCs). Expansion of PIGA and HLA class I mutant HSPCs have been linked to immune evasion from T-cell mediated pressures. We hypothesized that in analogy with antitumor immunity, the pathophysiological cascade of immune escape in IAA is initiated by immunoediting pressures and culminates with mechanisms of clonal evolution characterized by hits in immune recognition and response genes. To that end, we studied the genetic and transcriptomic make-up of the antigen presentation complexes in a large cohort of patients with IAA and paroxysmal nocturnal hemoglobinuria (PNH) by using single-cell RNA, high throughput DNA sequencing and single nucleotide polymorphism (SNP)-array platforms. At disease onset, HSPCs displayed activation of selected HLA class I and II-restricted mechanisms, without extensive inhibition of immune checkpoint apparatus. Using a newly implemented bioinformatic framework we found that not only class I but also class II genes were often impaired by acquisition of genetic aberrations. We also demonstrated the presence of novel somatic alterations in immune genes possibly contributing to the evasion from the autoimmune T-cells. In contrast, these hits were absent in myeloid neoplasia. These aberrations were not mutually exclusive with PNH and did not correlate with the accumulation of myeloid-driver hits. Our findings shed light on the mechanisms of immune activation and escape in IAA and define alternative modes of clonal hematopoiesis.

Somatic compensation of inherited bone marrow failure.

Inherited bone marrow failure syndromes (IBMFS) are a heterogeneous group of genetic disorders characterized by insufficient blood cell production and increased risk of transformation to myeloid malignancies. While genetically diverse, IBMFS are collectively defined by a cell-intrinsic hematopoietic stem cell (HSC) fitness defect that impairs HSC self-renewal and hematopoietic differentiation. In IBMFS, HSCs frequently acquire mutations that improve cell fitness, a phenomenon known as somatic compensation. Somatic compensation can occur via distinct genetic processes such as loss of the germline mutation or somatic alterations in pathways affected by the disease-causing gene. While the clinical implications of somatic compensation in IBMFS remain to be fully discovered, understanding these mutational processes can help understand disease pathophysiology and may inform future diagnostic and therapeutic approaches. In this review, we highlight current understanding about somatic compensation in IBMFS.

Clonal dynamics of hematopoietic stem cell compartment in aplastic anemia.

Advances in molecular technologies accelerated investigations of the hematopoietic stem cell (HSC) compartment in aplastic anemia (AA). Initially, stem cell biology approaches indicated a profound depletion of HSC pool, while studies of paroxysmal nocturnal hemoglobinuria (PNH), X-chromosome inactivation and cytogenetics provided the first evidence for the presence of clonality in the context of a contracted HSC compartment. More recently, the introduction of deep NGS allowed a more precise assessment of clonal expansions in AA. NGS studies demonstrated that the acquisition of somatic defects, including mutations and copy number alterations, can occur prior to and without a strict consequence of a later evolution of post-AA myelodysplastic syndrome (MDS). Such mutant clones may simply correspond to clonal hematopoiesis of indeterminate potential (CHIP) prematurely uncovered by the depletion of polyclonal "normal" HSCs. However, clonal expansions may also correspond to adaptive responses extrinsically or intrinsically enhancing clonal fitness. These processes could lead to adaptive (clonal non-malignant) or maladaptive (post-AA MDS) attempts at reconstitution of hematopoiesis. The spectrum of molecular events provides many clues as to the evolutionary forces driving the pathogenesis of AA and also valuable lessons as to the function of normal and malignant hematopoiesis. This article summarizes current thinking, controversies and dilemmas in interpreting molecular data obtained from the studies of AA.

Upregulation of Checkpoint Ligand Programmed Death-Ligand 1 in Patients with Paroxysmal Nocturnal Hemoglobinuria Explained by Proximal Complement Activation.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hemolytic disease driven by impaired complement regulation. Mutations in genes encoding the enzymes that build the GPI anchors are causative, with somatic mutations in the PIG-A gene occurring most frequently. As a result, the important membrane-bound complement regulators CD55 and CD59 are missing on the affected hematopoietic stem cells and their progeny, rendering those cells vulnerable to complement attack. Immune escape mechanisms sparing affected PNH stem cells from removal are suspected in the PNH pathogenesis, but molecular mechanisms have not been elucidated. We hypothesized that exuberant complement activity in PNH results in enhanced immune checkpoint interactions, providing a molecular basis for the potential immune escape in PNH. In a series of PNH patients, we found increased expression levels of the checkpoint ligand programmed death-ligand 1 (PD-L1) on granulocytes and monocytes, as well as in the plasma of PNH patients. Mechanistically, we demonstrate that complement activation leading to the decoration of particles/cells with C3- and/or C4-opsonins increased PD-L1 expression on neutrophils and monocytes as shown for different in vitro models of classical or alternative pathway activation. We further establish in vitro that complement inhibition at the level of C3, but not C5, inhibits the alternative pathway-mediated upregulation of PD-L1 and show by means of soluble PD-L1 that this observation translates into the clinical situation when PNH patients are treated with either C3 or C5 inhibitors. Together, the presented data show that the checkpoint ligand PD-L1 is increased in PNH patients, which correlates with proximal complement activation.

Clinical characteristics of 512 eculizumab-naive paroxysmal nocturnal hemoglobinuria patients in China: a single-center observational study.

OBJECTIVES: With large patient population and complement inhibitors naïve background, the characteristics patients with paroxysmal nocturnal hemoglobinuria (PNH) in China have not been well studied, especially for different subtypes. METHODS: We retrospectively reviewed patients with complete data who visited Peking Union Medical College Hospital (PUMCH) from 2009 to 2019 and had been followed up for more than 2 years. RESULTS: Five hundred and twelve patients were enrolled including 56.3% males and 43.7% females. The median age at disease onset was 33 (9∼80) years. Most were aged 21∼40 years (50.6%). 52.1%, 46.3% and 1.6% of the patients had classic PNH, bone marrow failure (BMF)/PNH and subclinical PNH, respectively. Symptoms of classic PNH were associated with hemolysis, whereas bleeding was more common in BMF/PNH patients. Classic PNH had higher survival rate, larger PNH clone size, higher lactate dehydrogenase (LDH) level and lower ferritin level than BMF/PNH. Although the rate of thrombosis was similar in the classic PNH and BMF/PNH (P = 0.66), those with BMF/PNH had higher chance of renal impairment (P < 0.05). Immunosuppressive agents was more common use in BMF/PNH (P < 0.05), but glucocorticoids, iron supplements and anticoagulants were more common used in classic PNH (P < 0.05) patients. Less evolution to myeloid malignancies was observed in classic PNH than in BMF/PNH (P = 0.02). The major causes of deaths were thrombosis (29.6%), hemorrhage (18.5%) and infections (18.5%). CONCLUSION: Patients with classic PNH and BMF/PNH have different clinical profiles, and we described a more hemolytic features of PNH in China which might be improved with complement inhibitors.

Persistent imbalance, anti-apoptotic, and anti-inflammatory signature of circulating C-C chemokines and cytokines in patients with paroxysmal nocturnal hemoglobinuria.

OBJECTIVE: Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal non-malignant disease in which hematopoietic cell apoptosis may play an important pathophysiological role. Previous studies of the content of phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) indicated the possibility of remote transmission of anti-apoptotic signals between pathological and normal hematopoietic progenitors. METHODS: The study determined the plasma levels of beta chemokines and cytokines in N = 19 patients with PNH and 31 healthy controls. The research material was peripheral blood plasma (EDTA) stored at -80 °C until the test. Beta chemokine and cytokine concentrations were tested in duplicate with Bio-Plex Pro Human Cytokine Assay (Bio-Rad, Hercules, CA, USA) using a Luminex 200 flow cytometer and xPONENT software (Luminex Corporation, Austin, TX, USA). In peripheral blood CD34+ cells we tested the proportions of PI(3,4,5)P3+ and Annexin binding apoptotic phenotype using FC and phosflow. RESULTS: Compared to the control group, the PNH group showed a significant increase in the plasma concentration of some beta chemokines and cytokines, including MIP-1alpha/CCL3, eotaxin/CCL11, MCP1/CCL2, IL4 and G-CSF. In the group of PNH patients, a significant decrease in the concentration of some cytokines was also observed: RANTES/CCL5, MIP-1beta/CCL4, PDGF-BB and IL9. At the same time, the plasma concentrations of the chemokine IP-10/CXCL10 and the cytokines IFN-gamma, TNF, IL6 and IL10 showed no significant deviations from the values for the control group. Anti-apoptotic phenotype and phosphatidylinositol (3,4,5)-trisphosphate content in PNH clone of CD34+ cells were associated with the level of CCL3 and negatively associated with CCL5, CCL4, PDGF-BB and IL9. CONCLUSIONS: This data suggest the existence of apoptotic and PI(3,4,5)P3 imbalance in PNH CD34+ cells driven by anti-apoptotic cytokine biosignature in PNH. Plasma cytokines and intracellular enzymes that regulate the phosphoinositide pathways may become a therapeutic target in PNH.

Analysis of baseline characteristics, disease burden and long-term follow-up of 167 patients with Paroxysmal Nocturnal Hemoglobinuria at a single center in Brazil.

Paroxysmal nocturnal hemoglobinuria (PNH) can occur as a hemolytic form or small PNH clone found in a patient with bone marrow failure. METHODS: Describe Brazilian retrospective PNH cohort and identify the impact of disease burden on long-term follow-up. RESULTS: 167 patients, mean age at diagnosis 28.4 (7.1-71.2 years), four years mean interval between onset of cytopenia/aplasia diagnosis and PNH clone detection. Patients were divided into 15 Classic PNH, 55 Hemolytic PNH with bone marrow hypoplasia (PNH/AA), and 97 Subclinical PNH (sc-PNH). Hypocellular bone marrow was found in 89.2%; 55 had hemoglobinuria and 22 thrombosis during monitoring. WBC PNH clone correlated with RBC PNH clone, LDH and cytopenia. Subclinical patients had lower median lower RBC clone (2.0% vs 24.0% vs 57.8%) and WBC clone (11.7% vs 58.8% vs 81.2%) than PNH/AA and Classic PNH, respectively. PNH granulocyte clone was 89.1% in thrombotic patients. Ten-year overall survival 80.4% and mortality in transplanted patients 9.6%. Sepsis was mortality cause in subclinical PNH (16/18, 88.8%), and thrombosis in hemolytic PNH (11/13, 84.6%). CONCLUSION: Large PNH clones and LDH burden were associated with increased hemolysis and thrombosis risks, while young patients were associated with small PNH clones and subclinical form of the disease. Knowledge of the patient profile, the low risk associated with HSCT, and the use of long-term IST may be instrumental in the clinical management of PNH in restricted-resources countries.

Advances in the creation of animal models of paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a disease caused by a phosphatidylinositol glycan anchor biosynthesis class A (PIG-A) mutation in hematopoietic stem cells. There are three theories about the possible mechanism of the pathogenesis of PNH: immune escape, anti-apoptotic mechanism, and secondary gene mutation. There has been little gain in the knowledge regarding its pathogenesis during the last decade owing to the lack of representative cell lines and animal models. There have been recent reports about the successful creation of PNH mouse and PNH rhesus macaque models. The detection of glycosylphosphatidylinositol-anchor protein (GPI-AP)-deficient cells and/or fluorescently labeled variant of aerolysin (FLAER) test, estimation of erythrocyte life span, and hemolysis-related experiments demonstrated that these animal models of PNH had GPI-AP-deficient blood cells with shortened lifespans and increased sensitivity to complement-activated hemolysis. However, there were no clinical manifestations such as hemolysis and thrombosis in these animal models. This suggested that the PIG-A mutation is one of the several conditions required for PNH, but it alone is not enough to cause PNH.

Time and residual hematopoiesis are crucial for PNH clones escape in hepatitis-associated aplastic anemia.

The presence of paroxysmal nocturnal hemoglobinuria (PNH) clones in aplastic anemia (AA) suggests immunopathogenesis, but when and how PNH clones emerge and proliferate are unclear. Hepatitis-associated aplastic anemia (HAAA) is a special variant of AA, contrarily to idiopathic AA, in HAAA the trigger for immune activation is clearer and represented by the hepatitis and thus serves as a good model for studying PNH clones. Ninety HAAA patients were enrolled, including 61 males and 29 females (median age 21 years). Four hundred three of idiopathic AA have been included as controls. The median time from hepatitis to cytopenia was 50 days (range 0-180 days) and from cytopenia to AA diagnosis was 26 days (range 2-370 days). PNH clones were detected in 8 HAAA patients (8.9%) at diagnosis and in 73 patients with idiopathic AA (IAA) (18.1%). PNH cells accounted for 4.2% (1.09-12.33%) of red cells and/or granulocytes and were more likely to be detected in patients with longer disease history and less severe disease. During follow-up, the cumulative incidence of PNH clones in HAAA increased to 18.9% (17/90). Nine HAAA patients newly developed PNH clones, including six immunosuppressive therapy (IST) nonresponders. The clone size was mostly stable during follow-up, and only 2 of 14 patients showed increased clone size without proof of hemolysis. In conclusion, PNH clones were infrequent in newly diagnosed HAAA, but their frequency increased to one that was similar to the IAA frequency during follow-up. These results suggest that the PNH clone selection/expansion process is dynamic and takes time to establish, confirming that retesting for PNH clones during follow-up is crucial.

Clinical and prognostic significance of small paroxysmal nocturnal hemoglobinuria clones in myelodysplastic syndrome and aplastic anemia.

In this large single-centre study, we report high prevalence (25%) of, small (<10%) and very small (<1%), paroxysmal nocturnal hemoglobinuria (PNH) clones by high-sensitive cytometry among 3085 patients tested. Given PNH association with bone marrow failures, we analyzed 869 myelodysplastic syndromes (MDS) and 531 aplastic anemia (AA) within the cohort. PNH clones were more frequent and larger in AA vs. MDS (p = 0.04). PNH clone, irrespective of size, was a good predictor of response to immunosuppressive therapy (IST) and to stem cell transplant (HSCT) (in MDS: 84% if PNH+ vs. 44.7% if PNH-, p = 0.01 for IST, and 71% if PNH+ vs. 56.6% if PNH- for HSCT; in AA: 78 vs. 50% for IST, p < 0.0001, and 97 vs. 77%, p = 0.01 for HSCT). PNH positivity had a favorable impact on disease progression (0.6% vs. 4.9% IPSS-progression in MDS, p < 0.005; and 2.1 vs. 6.9% progression to MDS in AA, p = 0.01), leukemic evolution (6.8 vs. 12.7%, p = 0.01 in MDS), and overall survival [73% (95% CI 68-77) vs. 51% (48-54), p < 0.0001], with a relative HR for mortality of 2.37 (95% CI 1.8-3.1; p < 0.0001) in PNH negative cases, both in univariate and multivariable analysis. Our data suggest systematic PNH testing in AA/MDS, as it might allow better prediction/prognostication and consequent clinical/laboratory follow-up timing.