Modelling and drug targeting of a myeloid neoplasm with atypical 3q26/MECOM rearrangement using patient-specific iPSCs.

Structural variations involving enhancer hijacking induce aberrant oncogene expression and cause tumorigenesis. A rare translocation, t(3;8)(q26.2;q24), is associated with MECOM and MYC rearrangement, causing myeloid neoplasms with a dismal prognosis. The most recent World Health Organization classification recognises myeloid neoplasms with MECOM rearrangement as acute myeloid leukaemia (AML) with defining genetic abnormalities. Recently, the increasing use of induced pluripotent stem cell (iPSC) technology has helped elucidate the pathogenic processes of haematological malignancies. However, its utility for investigating enhancer hijacking in myeloid neoplasms remains unclear. In this study, we generated iPSC lines from patients with myelodysplastic syndromes (MDS) harbouring t(3;8)(q26.2;q24) and differentiated them into haematopoietic progenitor cells to model the pathophysiology of MDS with t(3;8)(q26.2;q24). Our iPSC model reproduced the primary patient's MECOM expression changes and histone H3 lysine 27 acetylation (H3K27ac) patterns in the MECOM promoter and MYC blood enhancer cluster (BENC). Furthermore, we revealed the apoptotic effects of the bromodomain and extra-terminal motif (BET) inhibitor on iPSC-derived MDS cells by suppressing activated MECOM. Our study demonstrates the usefulness of iPSC models for uncovering the precise mechanism of enhancer hijacking due to chromosomal structural changes and discovering potential therapeutic drug candidates for cancer treatment.

[Concentration of gilteritinib in the cerebrospinal fluid of a patient with relapsed FLT3-ITD positive acute myeloid leukemia with optic nerve involvement].

A 72-year-old woman with relapsed FLT3-ITD-positive acute myeloid leukemia was treated with gilteritinib and achieved complete remission with incomplete hematological recovery. However, two months later, she developed optic nerve infiltration and lost vision in her right eye while maintaining hematological remission on gilteritinib. Intrathecal injection of cytotoxic drugs reduced the number of blasts in the cerebrospinal fluid (CSF), but her vision did not recover. At the onset of optic nerve infiltration, at a dose of 80 mg/day gilteritinib, the plasma trough and CSF levels of gilteritinib were 151.9 ng/ml and 1.9 ng/ml, respectively, with a central nervous system (CNS) penetration rate of 1.3%. Hematologic progressive disease (PD) was detected after 40 days, and the patient died one month later. Target sequencing at the time of hematologic PD revealed the FLT3 F691L mutation, which is known to confer resistance to gilteritinib. In this patient, pharmacokinetic (low CNS penetration of gilteritinib) and pharmacodynamic (acquisition of a drug resistance mutation) mechanisms were thought to be responsible for the CNS relapse and hematologic PD, respectively. We believe this is a valuable case to report considering the scarcity of data on CNS penetration of FLT3 inhibitors and their effects on CNS disease in the literature.

Juvenile Hemochromatosis With Non-transfused Hemolytic Anemia Caused by a De Novo PIEZO1 Gene Mutation.

Differential diagnosis of juvenile hemochromatosis along with hemolytic anemia is often difficult. We report a 23-year-old woman with macrocytic hemolytic anemia with iron overload. The patient showed high serum ferritin and transferrin saturation and low serum transferrin and ceruloplasmin. We also noticed stomatocytes in her blood smear, which was confirmed by scanning electron microscopy. Target gene sequencing identified a mutation in PIEZO1 (heterozygous c.6008C>A: p.A2003D). This mutation was reported previously in a family with dehydrated hereditary stomatocytosis (DHS1, [OMIM 194380]), but in the current case, it was identified to be a de novo mutation. We underscore DHS1 in the differential diagnosis of iron overload associated with non-transfused hemolytic anemia in children and young adults.

A frequent nonsense mutation in exon 1 across certain HLA-A and -B alleles in leukocytes of patients with acquired aplastic anemia.

Leukocytes that lack HLA allelic expression are frequently detected in patients with acquired aplastic anemia (AA) who respond to immunosuppressive therapy (IST), although the exact mechanisms underlying the HLA loss and HLA allele repertoire likely to acquire loss-of-function mutations are unknown. We identified a common nonsense mutation at position 19 (c.19C>T, p.R7X) in exon 1 (Exon1mut) of different HLA-A and -B alleles in HLA-lacking granulocytes from AA patients. A droplet digital PCR (ddPCR) assay capable of detecting as few as 0.07% Exon1mut HLA alleles in total DNA revealed the mutation was present in 29% (101/353) of AA patients, with a median allele frequency of 0.42% (range, 0.071% to 21.3%). Exon1mut occurred in only 12 different HLA-A (n=4) and HLA-B (n=8) alleles, including B*40:02 (n=31) and A*02:06 (n=15), which correspond to 4 HLA supertypes (A02, A03, B07, and B44). The percentages of patients who possessed at least one of these 12 HLA alleles were significantly higher in the 353 AA patients (92%, P.

Allele-specific ablation rescues electrophysiological abnormalities in a human iPS cell model of long-QT syndrome with a CALM2 mutation.

Calmodulin is a ubiquitous Ca2+ sensor molecule encoded by three distinct calmodulin genes, CALM1-3. Recently, mutations in CALM1-3 have been reported to be associated with severe early-onset long-QT syndrome (LQTS). However, the underlying mechanism through which heterozygous calmodulin mutations lead to severe LQTS remains unknown, particularly in human cardiomyocytes. We aimed to establish an LQTS disease model associated with a CALM2 mutation (LQT15) using human induced pluripotent stem cells (hiPSCs) and to assess mutant allele-specific ablation by genome editing for the treatment of LQT15. We generated LQT15-hiPSCs from a 12-year-old boy with LQTS carrying a CALM2-N98S mutation and differentiated these hiPSCs into cardiomyocytes (LQT15-hiPSC-CMs). Action potentials (APs) and L-type Ca2+ channel (LTCC) currents in hiPSC-CMs were analyzed by the patch-clamp technique and compared with those of healthy controls. Furthermore, we performed mutant allele-specific knockout using a CRISPR-Cas9 system and analyzed electrophysiological properties. Electrophysiological analyses revealed that LQT15-hiPSC-CMs exhibited significantly lower beating rates, prolonged AP durations, and impaired inactivation of LTCC currents compared with control cells, consistent with clinical phenotypes. Notably, ablation of the mutant allele rescued the electrophysiological abnormalities of LQT15-hiPSC-CMs, indicating that the mutant allele caused dominant-negative suppression of LTCC inactivation, resulting in prolonged AP duration. We successfully recapitulated the disease phenotypes of LQT15 and revealed that inactivation of LTCC currents was impaired in CALM2-N98S hiPSC model. Additionally, allele-specific ablation using the latest genome-editing technology provided important insights into a promising therapeutic approach for inherited cardiac diseases.

Development of a Patient-Derived Induced Pluripotent Stem Cell Model for the Investigation of SCN5A-D1275N-Related Cardiac Sodium Channelopathy.

BACKGROUND: TheSCN5Agene encodes the α subunit of the cardiac voltage-gated sodium channel, NaV1.5. The missense mutation, D1275N, has been associated with a range of unusual phenotypes associated with reduced NaV1.5 function, including cardiac conduction disease and dilated cardiomyopathy. Curiously, the reported biophysical properties ofSCN5A-D1275N channels vary with experimental system.Methods and Results:First, using a human embryonic kidney (HEK) 293 cell-based heterologous expression system, theSCN5A-D1275N channels showed similar maximum sodium conductance but a significantly depolarizing shift of activation gate (+10 mV) compared to wild type. Second, we generated human-induced pluripotent stem cells (hiPSCs) from a 24-year-old female who carried heterozygousSCN5A-D1275N and analyzed the differentiated cardiomyocytes (CMs). AlthoughSCN5Atranscript levels were equivalent between D1275N and control hiPSC-CMs, both the total amount of NaV1.5 and the membrane fractions were reduced approximately half in the D1275N cells, which were rescued by the proteasome inhibitor MG132 treatment. Electrophysiological assays revealed that maximum sodium conductance was reduced to approximately half of that in control hiPSC-CMs in the D1275N cells, and maximum upstroke velocity of action potential was lower in D1275N, which was consistent with the reduced protein level of NaV1.5. CONCLUSIONS: This study successfully demonstrated diminished sodium currents resulting from lower NaV1.5 protein levels, which is dependent on proteasomal degradation, using a hiPSC-based model forSCN5A-D1275N-related sodium channelopathy.

Reprogramming technology reveals genetic and functional diversity of subclones in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal stem cell diseases characterized by inefficient hematopoiesis and poor prognosis. There are currently no useful tools for identifying new therapeutic targets of MDS mainly because of a lack of good disease models. Although massive parallel sequencing studies have revealed several MDS-specific genomic alterations that are different from those of de novo acute myeloid leukemia (AML), the relationships between the genetic architecture and pathophysiology in MDS remain poorly understood. We successfully generated multiple iPS cell lines (MDS-iPSC lines) from several patients with either MDS or secondary AML that progressed from MDS. We assessed the hematopoietic differentiation potential of the established MDS-iPSC lines and identified stage-specific maturation defects with graded severity. The MDS-iPSC lines could be a useful tool for elucidating the subclonal diversity, pathogenesis, and clonal evolution of MDS as well as for identifying new therapeutic compounds.

CD151 expression marks atrial- and ventricular- differentiation from human induced pluripotent stem cells.

Current differentiation protocols for human induced pluripotent stem cells (hiPSCs) produce heterogeneous cardiomyocytes (CMs). Although chamber-specific CM selection using cell surface antigens enhances biomedical applications, a cell surface marker that accurately distinguishes between hiPSC-derived atrial CMs (ACMs) and ventricular CMs (VCMs) has not yet been identified. We have developed an approach for obtaining functional hiPSC-ACMs and -VCMs based on CD151 expression. For ACM differentiation, we found that ACMs are enriched in the CD151low population and that CD151 expression is correlated with the expression of Notch4 and its ligands. Furthermore, Notch signaling inhibition followed by selecting the CD151low population during atrial differentiation leads to the highly efficient generation of ACMs as evidenced by gene expression and electrophysiology. In contrast, for VCM differentiation, VCMs exhibiting a ventricular-related gene signature and uniform action potentials are enriched in the CD151high population. Our findings enable the production of high-quality ACMs and VCMs appropriate for hiPSC-derived chamber-specific disease models and other applications.

Disrupted CaV1.2 selectivity causes overlapping long QT and Brugada syndrome phenotypes in the CACNA1C-E1115K iPS cell model.

BACKGROUND: A missense mutation in the α1c subunit of voltage-gated L-type Ca2+ channel-coding CACNA1C-E1115K, located in the Ca2+ selectivity site, causes a variety of arrhythmogenic phenotypes. OBJECTIVE: We aimed to investigate the electrophysiological features and pathophysiological mechanisms of CACNA1C-E1115K in patient-specific induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs). METHODS: We generated iPSCs from a patient carrying heterozygous CACNA1C-E1115K with overlapping phenotypes of long QT syndrome, Brugada syndrome, and mild cardiac dysfunction. Electrophysiological properties were investigated using iPSC-CMs. We used iPSCs from a healthy individual and an isogenic iPSC line corrected using CRISPR-Cas9-mediated gene editing as controls. A mathematical E1115K-CM model was developed using a human ventricular cell model. RESULTS: Patch-clamp analysis revealed that E1115K-iPSC-CMs exhibited reduced peak Ca2+ current density and impaired Ca2+ selectivity with an increased permeability to monovalent cations. Consequently, E1115K-iPSC-CMs showed decreased action potential plateau amplitude, longer action potential duration (APD), and a higher frequency of early afterdepolarization compared with controls. In optical recordings examining the antiarrhythmic drug effect, late Na+ channel current (INaL) inhibitors (mexiletine and GS-458967) shortened APDs specifically in E1115K-iPSC-CMs. The AP-clamp using a voltage command obtained from E1115K-iPSC-CMs with lower action potential plateau amplitude and longer APD confirmed the upregulation of INaL. An in silico study recapitulated the in vitro electrophysiological properties. CONCLUSION: Our iPSC-based analysis in CACNA1C-E1115K with disrupted CaV1.2 selectivity demonstrated that the aberrant currents through the mutant channels carried by monovalent cations resulted in specific action potential changes, which increased endogenous INaL, thereby synergistically contributing to the arrhythmogenic phenotype.

Novel Calmodulin Variant p.E46K Associated With Severe Catecholaminergic Polymorphic Ventricular Tachycardia Produces Robust Arrhythmogenicity in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

BACKGROUND: CaM (calmodulin) is a ubiquitously expressed, multifunctional Ca2+ sensor protein that regulates numerous proteins. Recently, CaM missense variants have been identified in patients with malignant inherited arrhythmias, such as long QT syndrome and catecholaminergic polymorphic ventricular tachycardia (CPVT). However, the exact mechanism of CaM-related CPVT in human cardiomyocytes remains unclear. In this study, we sought to investigate the arrhythmogenic mechanism of CPVT caused by a novel variant using human induced pluripotent stem cell (iPSC) models and biochemical assays. METHODS: We generated iPSCs from a patient with CPVT bearing CALM2 p.E46K. As comparisons, we used 2 control lines including an isogenic line, and another iPSC line from a patient with long QT syndrome bearing CALM2 p.N98S (also reported in CPVT). Electrophysiological properties were investigated using iPSC-cardiomyocytes. We further examined the RyR2 (ryanodine receptor 2) and Ca2+ affinities of CaM using recombinant proteins. RESULTS: We identified a novel de novo heterozygous variant, CALM2 p.E46K, in 2 unrelated patients with CPVT accompanied by neurodevelopmental disorders. The E46K-cardiomyocytes exhibited more frequent abnormal electrical excitations and Ca2+ waves than the other lines in association with increased Ca2+ leakage from the sarcoplasmic reticulum via RyR2. Furthermore, the [3H]ryanodine binding assay revealed that E46K-CaM facilitated RyR2 function especially by activating at low [Ca2+] levels. The real-time CaM-RyR2 binding analysis demonstrated that E46K-CaM had a 10-fold increased RyR2 binding affinity compared with wild-type CaM which may account for the dominant effect of the mutant CaM. Additionally, the E46K-CaM did not affect CaM-Ca2+ binding or L-type calcium channel function. Finally, antiarrhythmic agents, nadolol and flecainide, suppressed abnormal Ca2+ waves in E46K-cardiomyocytes. CONCLUSIONS: We, for the first time, established a CaM-related CPVT iPSC-CM model which recapitulated severe arrhythmogenic features resulting from E46K-CaM dominantly binding and facilitating RyR2. In addition, the findings in iPSC-based drug testing will contribute to precision medicine.

Purification of human iPSC-derived cells at large scale using microRNA switch and magnetic-activated cell sorting.

For regenerative cell therapies using pluripotent stem cell (PSC)-derived cells, large quantities of purified cells are required. Magnetic-activated cell sorting (MACS) is a powerful approach to collect target antigen-positive cells; however, it remains a challenge to purify various cell types efficiently at large scale without using antibodies specific to the desired cell type. Here we develop a technology that combines microRNA (miRNA)-responsive mRNA switch (miR-switch) with MACS (miR-switch-MACS) to purify large amounts of PSC-derived cells rapidly and effectively. We designed miR-switches that detect specific miRNAs expressed in target cells and controlled the translation of a CD4-coding transgene as a selection marker for MACS. For the large-scale purification of induced PSC-derived cardiomyocytes (iPSC-CMs), we transferred miR-208a-CD4 switch-MACS and obtained purified iPSC-CMs efficiently. Moreover, miR-375-CD4 switch-MACS highly purified pancreatic insulin-producing cells and their progenitors expressing Chromogranin A. Overall, the miR-switch-MACS method can efficiently purify target PSC-derived cells for cell replacement therapy.

Azacitidine is a potential therapeutic drug for pyridoxine-refractory female X-linked sideroblastic anemia.

X-linked sideroblastic anemia (XLSA) is associated with mutations in the erythroid-specific δ-aminolevulinic acid synthase (ALAS2) gene. Treatment of XLSA is mainly supportive, except in patients who are pyridoxine responsive. Female XLSA often represents a late onset of severe anemia, mostly related to the acquired skewing of X chromosome inactivation. In this study, we successfully generated active wild-type and mutant ALAS2-induced pluripotent stem cell (iPSC) lines from the peripheral blood cells of an affected mother and 2 daughters in a family with pyridoxine-resistant XLSA related to a heterozygous ALAS2 missense mutation (R227C). The erythroid differentiation potential was severely impaired in active mutant iPSC lines compared with that in active wild-type iPSC lines. Most of the active mutant iPSC-derived erythroblasts revealed an immature morphological phenotype, and some showed dysplasia and perinuclear iron deposits. In addition, globin and HO-1 expression and heme biosynthesis in active mutant erythroblasts were severely impaired compared with that in active wild-type erythroblasts. Furthermore, genes associated with erythroblast maturation and karyopyknosis showed significantly reduced expression in active mutant erythroblasts, recapitulating the maturation defects. Notably, the erythroid differentiation ability and hemoglobin expression of active mutant iPSC-derived hematopoietic progenitor cells (HPCs) were improved by the administration of δ-aminolevulinic acid, verifying the suitability of the cells for drug testing. Administration of a DNA demethylating agent, azacitidine, reactivated the silent, wild-type ALAS2 allele in active mutant HPCs and ameliorated the erythroid differentiation defects, suggesting that azacitidine is a potential novel therapeutic drug for female XLSA. Our patient-specific iPSC platform provides novel biological and therapeutic insights for XLSA.

Hematopoietic stem progenitor cells with malignancy-related gene mutations in patients with acquired aplastic anemia are characterized by the increased expression of CXCR4.

The phenotypic changes in hematopoietic stem progenitor cells (HSPCs) with somatic mutations of malignancy-related genes in patients with acquired aplastic anemia (AA) are poorly understood. As our initial study showed increased CXCR4 expression on HLA allele-lacking (HLA[-]) HSPCs that solely support hematopoiesis in comparison to redundant HLA(+) HSPCs in AA patients, we screened the HSPCs of patients with various types of bone marrow (BM) failure to investigate their CXCR4 expression. In comparison to healthy individuals (n = 15, 12.3%-49.9%, median 43.2%), the median CXCR4+ cell percentages in the HSPCs of patients without somatic mutations were low: 29.3% (14.3%-37.3%) in the eight patients without HLA(-) granulocytes, 8.8% (4.1%-9.8%) in the five patients with HLA(-) cells accounting for >90% of granulocytes, and 7.8 (2.1%-8.7%) in the six patients with paroxysmal nocturnal hemoglobinuria. In contrast, the median percentage was much higher (78% [61.4%-88.7%]) in the five AA patients without HLA(-) granulocytes possessing somatic mutations (c-kit, t[8;21], monosomy 7 [one for each], ASXL1 [n = 2]), findings that were comparable to those (66.5%, 63.1%-88.9%) in the four patients with advanced myelodysplastic syndromes. The increased expression of CXCR4 may therefore reflect intrinsic abnormalities of HSPCs caused by somatic mutations that allow them to evade restriction by BM stromal cells.

The GPI-anchored protein CD109 protects hematopoietic progenitor cells from undergoing erythroid differentiation induced by TGF-ß.

Although a glycosylphosphatidylinositol-anchored protein (GPI-AP) CD109 serves as a TGF-ß co-receptor and inhibits TGF-ß signaling in keratinocytes, the role of CD109 on hematopoietic stem progenitor cells (HSPCs) remains unknown. We studied the effect of CD109 knockout (KO) or knockdown (KD) on TF-1, a myeloid leukemia cell line that expresses CD109, and primary human HSPCs. CD109-KO or KD TF-1 cells underwent erythroid differentiation in the presence of TGF-ß. CD109 was more abundantly expressed in hematopoietic stem cells (HSCs) than in multipotent progenitors and HSPCs of human bone marrow (BM) and cord blood but was not detected in mouse HSCs. Erythroid differentiation was induced by TGF-ß to a greater extent in CD109-KD cord blood or iPS cell-derived megakaryocyte-erythrocyte progenitor cells (MEPs) than in wild-type MEPs. When we analyzed the phenotype of peripheral blood MEPs of patients with paroxysmal nocturnal hemoglobinuria who had both GPI(+) and GPI(-) CD34+ cells, the CD36 expression was more evident in CD109- MEPs than CD109+ MEPs. In summary, CD109 suppresses TGF-ß signaling in HSPCs, and the lack of CD109 may increase the sensitivity of PIGA-mutated HSPCs to TGF-ß, thus leading to the preferential commitment of erythroid progenitor cells to mature red blood cells in immune-mediated BM failure.

Kpm/Lats2 is linked to chemosensitivity of leukemic cells through the stabilization of p73.

Down-regulation of the Kpm/Lats2 tumor suppressor is observed in various malignancies and associated with poor prognosis in acute lymphoblastic leukemia. We documented that Kpm/Lats2 was markedly decreased in several leukemias that were highly resistant to conventional chemotherapy. Silencing of Kpm/Lats2 expression in leukemic cells did not change the rate of cell growth but rendered the cells more resistant to DNA damage-inducing agents. Expression of p21 and PUMA was strongly induced by these agents in control cells, despite defective p53, but was only slightly induced in Kpm/Lats2-knockdown cells. DNA damage-induced nuclear accumulation of p73 was clearly observed in control cells but hardly detected in Kpm/Lats2-knockdown cells. Chromatin immunoprecipitation (ChIP) assay showed that p73 was recruited to the PUMA gene promoter in control cells but not in Kpm/Lats2-knockdown cells after DNA damage. The analyses with transient coexpression of Kpm/Lats2, YAP2, and p73 showed that Kpm/Lats2 contributed the stability of YAP2 and p73, which was dependent on the kinase function of Kpm/Lats2 and YAP2 phosphorylation at serine 127. Our results suggest that Kpm/Lats2 is involved in the fate of p73 through the phosphorylation of YAP2 by Kpm/Lats2 and the induction of p73 target genes that underlie chemosensitivity of leukemic cells.

Erratum to "Optical Recording of Action Potentials in Human Induced Pluripotent Stem Cell-Derived Cardiac Single Cells and Monolayers Generated from Long QT Syndrome Type 1 Patients".

[This corrects the article DOI: 10.1155/2019/7532657.].