Expansion effect of romiplostim on hematopoietic stem and progenitor cells versus thrombopoietin and eltrombopag.

Romiplostim, a thrombopoietin (TPO) receptor agonist, is a clinically approved drug that is clearly effective in reconstituting hematopoiesis in refractory aplastic anemia and　idiopathic thrombocytopenic purpura. However, the mechanism underlying its biological effect is unknown, and its differences from other TPO receptor agonists remain unclear. Therefore, we determined the in vitro expansion effect of romiplostim on human CD34 + hematopoietic stem and progenitor cells (HSPCs) versus recombinant human TPO (rhTPO) and another clinically available drug, eltrombopag. We also performed single-cell RNA-seq to determine effects of romiplostim on CD34 + HSPCs at the molecular level. The maximum expansion effect of romiplostim on total CD34 + cells, CD34 + CD38 + progenitor cells, and CD34 + CD38 - immature cells was comparable to that of rhTPO, but higher than that of eltrombopag, particularly on CD34 + CD38 - immature cells. Single-cell RNA-seq analysis revealed that both romiplostim and eltrombopag induced signatures driven by rhTPO, but romiplostim induced molecular changes related to RHOA signaling in the most primitive HSPC subsets that were partially driven or not driven by eltrombopag. Additionally, romiplostim did not induce TFRC expression as was observed with eltrombopag. In conclusion, romiplostim expands and affects human HSPCs similar to rhTPO, but partially different from eltrombopag in terms of induction of gene expression.

Phospholipid metabolic adaptation promotes survival of IDH2 mutant acute myeloid leukemia cells.

Genetic mutations in the isocitrate dehydrogenase (IDH) gene that result in a pathological enzymatic activity to produce oncometabolite have been detected in acute myeloid leukemia (AML) patients. While specific inhibitors that target mutant IDH enzymes and normalize intracellular oncometabolite level have been developed, refractoriness and resistance has been reported. Since acquisition of pathological enzymatic activity is accompanied by the abrogation of the crucial WT IDH enzymatic activity in IDH mutant cells, aberrant metabolism in IDH mutant cells can potentially persist even after the normalization of intracellular oncometabolite level. Comparisons of isogenic AML cell lines with and without IDH2 gene mutations revealed two mutually exclusive signalings for growth advantage of IDH2 mutant cells, STAT phosphorylation associated with intracellular oncometabolite level and phospholipid metabolic adaptation. The latter came to light after the oncometabolite normalization and increased the resistance of IDH2 mutant cells to arachidonic acid-mediated apoptosis. The release of this metabolic adaptation by FDA-approved anti-inflammatory drugs targeting the metabolism of arachidonic acid could sensitize IDH2 mutant cells to apoptosis, resulting in their eradication in vitro and in vivo. Our findings will contribute to the development of alternative therapeutic options for IDH2 mutant AML patients who do not tolerate currently available therapies.

Akkermansia muciniphila induces slow extramedullary hematopoiesis via cooperative IL-1R/TLR signals.

Bacterial infections can activate and mobilize hematopoietic stem and progenitor cells (HSPCs) from the bone marrow (BM) to the spleen, a process termed extramedullary hematopoiesis (EMH). Recent studies suggest that commensal bacteria regulate not only the host immune system but also hematopoietic homeostasis. However, the impact of gut microbes on hematopoietic pathology remains unclear. Here, we find that systemic single injections of Akkermansia muciniphila (A. m.), a mucin-degrading bacterium, rapidly activate BM myelopoiesis and slow but long-lasting hepato-splenomegaly, characterized by the expansion and differentiation of functional HSPCs, which we term delayed EMH. Mechanistically, delayed EMH triggered by A. m. is mediated entirely by the MYD88/TRIF innate immune signaling pathway, which persistently stimulates splenic myeloid cells to secrete interleukin (IL)-1α, and in turn, activates IL-1 receptor (IL-1R)-expressing splenic HSPCs. Genetic deletion of Toll-like receptor-2 and -4 (TLR2/4) or IL-1α partially diminishes A. m.-induced delayed EMH, while inhibition of both pathways alleviates splenomegaly and EMH. Our results demonstrate that cooperative IL-1R- and TLR-mediated signals regulate commensal bacteria-driven EMH, which might be relevant for certain autoimmune disorders.

GPRC5C drives branched-chain amino acid metabolism in leukemogenesis.

Leukemia stem cells (LSCs) share numerous features with healthy hematopoietic stem cells (HSCs). G-protein coupled receptor family C group 5 member C (GPRC5C) is a regulator of HSC dormancy. However, GPRC5C functionality in acute myeloid leukemia (AML) is yet to be determined. Within patient AML cohorts, high GPRC5C levels correlated with poorer survival. Ectopic Gprc5c expression increased AML aggression through the activation of NF-κB, which resulted in an altered metabolic state with increased levels of intracellular branched-chain amino acids (BCAAs). This onco-metabolic profile was reversed upon loss of Gprc5c, which also abrogated the leukemia-initiating potential. Targeting the BCAA transporter SLC7A5 with JPH203 inhibited oxidative phosphorylation and elicited strong antileukemia effects, specifically in mouse and patient AML samples while sparing healthy bone marrow cells. This antileukemia effect was strengthened in the presence of venetoclax and azacitidine. Our results indicate that the GPRC5C-NF-κB-SLC7A5-BCAAs axis is a therapeutic target that can compromise leukemia stem cell function in AML.

Humanized mouse models with endogenously developed human natural killer cells for in vivo immunogenicity testing of HLA class I-edited iPSC-derived cells.

Human induced pluripotent stem cells (hiPSCs) genetically depleted of human leucocyte antigen (HLA) class I expression can bypass T cell alloimmunity and thus serve as a one-for-all source for cell therapies. However, these same therapies may elicit rejection by natural killer (NK) cells, since HLA class I molecules serve as inhibitory ligands of NK cells. Here, we focused on testing the capacity of endogenously developed human NK cells in humanized mice (hu-mice) using MTSRG and NSG-SGM3 strains to assay the tolerance of HLA-edited iPSC-derived cells. High NK cell reconstitution was achieved with the engraftment of cord blood-derived human hematopoietic stem cells (hHSCs) followed by the administration of human interleukin-15 (hIL-15) and IL-15 receptor alpha (hIL-15Rα). Such "hu-NK mice" rejected HLA class I-null hiPSC-derived hematopoietic progenitor cells (HPCs), megakaryocytes and T cells, but not HLA-A/B-knockout, HLA-C expressing HPCs. To our knowledge, this study is the first to recapitulate the potent endogenous NK cell response to non-tumor HLA class I-downregulated cells in vivo. Our hu-NK mouse models are suitable for the non-clinical evaluation of HLA-edited cells and will contribute to the development of universal off-the-shelf regenerative medicine.

Hematopoietic stem and progenitor cells integrate microbial signals to promote post-inflammation gut tissue repair.

Bone marrow (BM)-resident hematopoietic stem and progenitor cells (HSPCs) are often activated following bacterial insults to replenish the host hemato-immune system, but how they integrate the associated tissue damage signals to initiate distal tissue repair is largely unknown. Here, we show that acute gut inflammation expands HSPCs in the BM and directs them to inflamed mesenteric lymph nodes through GM-CSFR activation for further expansion and potential differentiation into Ly6C+ /G+ myeloid cells specialized in gut tissue repair. We identified this process to be mediated by Bacteroides, a commensal gram-negative bacteria that activates innate immune signaling. These findings establish cross-organ communication between the BM and distant inflamed sites, whereby a certain subset of multipotent progenitors is specified to respond to imminent hematopoietic demands and to alleviate inflammatory symptoms.

Unique molecular and functional features of extramedullary hematopoietic stem and progenitor cell reservoirs in humans.

Rare hematopoietic stem and progenitor cell (HSPC) pools outside the bone marrow (BM) contribute to blood production in stress and disease but remain ill-defined. Although nonmobilized peripheral blood (PB) is routinely sampled for clinical management, the diagnosis and monitoring potential of PB HSPCs remain untapped, as no healthy PB HSPC baseline has been reported. Here we comprehensively delineate human extramedullary HSPC compartments comparing spleen, PB, and mobilized PB to BM using single-cell RNA-sequencing and/or functional assays. We uncovered HSPC features shared by extramedullary tissues and others unique to PB. First, in contrast to actively dividing BM HSPCs, we found no evidence of substantial ongoing hematopoiesis in extramedullary tissues at steady state but report increased splenic HSPC proliferative output during stress erythropoiesis. Second, extramedullary hematopoietic stem cells/multipotent progenitors (HSCs/MPPs) from spleen, PB, and mobilized PB share a common transcriptional signature and increased abundance of lineage-primed subsets compared with BM. Third, healthy PB HSPCs display a unique bias toward erythroid-megakaryocytic differentiation. At the HSC/MPP level, this is functionally imparted by a subset of phenotypic CD71+ HSCs/MPPs, exclusively producing erythrocytes and megakaryocytes, highly abundant in PB but rare in other adult tissues. Finally, the unique erythroid-megakaryocytic-skewing of PB is perturbed with age in essential thrombocythemia and ß-thalassemia. Collectively, we identify extramedullary lineage-primed HSPC reservoirs that are nonproliferative in situ and report involvement of splenic HSPCs during demand-adapted hematopoiesis. Our data also establish aberrant composition and function of circulating HSPCs as potential clinical indicators of BM dysfunction.

Fit-For-All iPSC-Derived Cell Therapies and Their Evaluation in Humanized Mice With NK Cell Immunity.

Human induced pluripotent stem cells (iPSCs) can be limitlessly expanded and differentiated into almost all cell types. Moreover, they are amenable to gene manipulation and, because they are established from somatic cells, can be established from essentially any person. Based on these characteristics, iPSCs have been extensively studied as cell sources for tissue grafts, blood transfusions and cancer immunotherapies, and related clinical trials have started. From an immune-matching perspective, autologous iPSCs are perfectly compatible in principle, but also require a prolonged time for reaching the final products, have high cost, and person-to-person variation hindering their common use. Therefore, certified iPSCs with reduced immunogenicity are expected to become off-the-shelf sources, such as those made from human leukocyte antigen (HLA)-homozygous individuals or genetically modified for HLA depletion. Preclinical tests using immunodeficient mice reconstituted with a human immune system (HIS) serve as an important tool to assess the human alloresponse against iPSC-derived cells. Especially, HIS mice reconstituted with not only human T cells but also human natural killer (NK) cells are considered crucial. NK cells attack so-called "missing self" cells that do not express self HLA class I, which include HLA-homozygous cells that express only one allele type and HLA-depleted cells. However, conventional HIS mice lack enough reconstituted human NK cells for these tests. Several measures have been developed to overcome this issue including the administration of cytokines that enhance NK cell expansion, such as IL-2 and IL-15, the administration of vectors that express those cytokines, and genetic manipulation to express the cytokines or to enhance the reconstitution of human myeloid cells that express IL15R-alpha. Using such HIS mice with enhanced human NK cell reconstitution, alloresponses against HLA-homozygous and HLA-depleted cells have been studied. However, most studies used HLA-downregulated tumor cells as the target cells and tested in vitro after purifying human cells from HIS mice. In this review, we give an overview of the current state of iPSCs in cell therapies, strategies to lessen their immunogenic potential, and then expound on the development of HIS mice with reconstituted NK cells, followed by their utilization in evaluating future universal HLA-engineered iPSC-derived cells.

iPSC modeling of stage-specific leukemogenesis reveals BAALC as a key oncogene in severe congenital neutropenia.

Severe congenital neutropenia (CN) is a pre-leukemic bone marrow failure syndrome that can evolve to acute myeloid leukemia (AML). Mutations in CSF3R and RUNX1 are frequently observed in CN patients, although how they drive the transition from CN to AML (CN/AML) is unclear. Here we establish a model of stepwise leukemogenesis in CN/AML using CRISPR-Cas9 gene editing of CN patient-derived iPSCs. We identified BAALC upregulation and resultant phosphorylation of MK2a as a key leukemogenic event. BAALC deletion or treatment with CMPD1, a selective inhibitor of MK2a phosphorylation, blocked proliferation and induced differentiation of primary CN/AML blasts and CN/AML iPSC-derived hematopoietic stem and progenitor cells (HSPCs) without affecting healthy donor or CN iPSC-derived HSPCs. Beyond detailing a useful method for future investigation of stepwise leukemogenesis, this study suggests that targeting BAALC and/or MK2a phosphorylation may prevent leukemogenic transformation or eliminate AML blasts in CN/AML and RUNX1 mutant BAALC(hi) de novo AML.

NAMPT/SIRT2-mediated inhibition of the p53-p21 signaling pathway is indispensable for maintenance and hematopoietic differentiation of human iPS cells.

BACKGROUND: Nicotinamide phosphoribosyltransferase (NAMPT) regulates cellular functions through the protein deacetylation activity of nicotinamide adenine dinucleotide (NAD+)-dependent sirtuins (SIRTs). SIRTs regulate functions of histones and none-histone proteins. The role of NAMPT/SIRT pathway in the regulation of maintenance and differentiation of human-induced pluripotent stem (iPS) cells is not fully elucidated. METHODS: We evaluated the effects of specific inhibitors of NAMPT or SIRT2 on the pluripotency, proliferation, survival, and hematopoietic differentiation of human iPS cells. We also studied the molecular mechanism downstream of NAMPT/SIRTs in iPS cells. RESULTS: We demonstrated that NAMPT is indispensable for the maintenance, survival, and hematopoietic differentiation of iPS cells. We found that inhibition of NAMPT or SIRT2 in iPS cells induces p53 protein by promoting its lysine acetylation. This leads to activation of the p53 target, p21, with subsequent cell cycle arrest and induction of apoptosis in iPS cells. NAMPT and SIRT2 inhibition also affect hematopoietic differentiation of iPS cells in an embryoid body (EB)-based cell culture system. CONCLUSIONS: Our data demonstrate the essential role of the NAMPT/SIRT2/p53/p21 signaling axis in the maintenance and hematopoietic differentiation of iPS cells.

LMO2 activation by deacetylation is indispensable for hematopoiesis and T-ALL leukemogenesis.

Hematopoietic transcription factor LIM domain only 2 (LMO2), a member of the TAL1 transcriptional complex, plays an essential role during early hematopoiesis and is frequently activated in T-cell acute lymphoblastic leukemia (T-ALL) patients. Here, we demonstrate that LMO2 is activated by deacetylation on lysine 74 and 78 via the nicotinamide phosphoribosyltransferase (NAMPT)/sirtuin 2 (SIRT2) pathway. LMO2 deacetylation enables LMO2 to interact with LIM domain binding 1 and activate the TAL1 complex. NAMPT/SIRT2-mediated activation of LMO2 by deacetylation appears to be important for hematopoietic differentiation of induced pluripotent stem cells and blood formation in zebrafish embryos. In T-ALL, deacetylated LMO2 induces expression of TAL1 complex target genes HHEX and NKX3.1 as well as LMO2 autoregulation. Consistent with this, inhibition of NAMPT or SIRT2 suppressed the in vitro growth and in vivo engraftment of T-ALL cells via diminished LMO2 deacetylation. This new molecular mechanism may provide new therapeutic possibilities in T-ALL and may contribute to the development of new methods for in vitro generation of blood cells.

Human iPSC-based model of severe congenital neutropenia reveals elevated UPR and DNA damage in CD34+ cells preceding leukemic transformation.

We describe the establishment of an embryoid-body-based protocol for hematopoietic/myeloid differentiation of human induced pluripotent stem cells that allows the generation of CD34+ cells or mature myeloid cells in vitro. Using this model, we were able to recapitulate the defective granulocytic differentiation in patients with severe congenital neutropenia (CN), an inherited preleukemia bone marrow failure syndrome. Importantly, in vitro maturation arrest of granulopoiesis was associated with an elevated unfolded protein response (UPR) and enhanced expression of the cell cycle inhibitor p21. Consistent with this, we found that CD34+ cells of CN patients were highly susceptible to DNA damage and showed diminished DNA repair. These observations suggest that targeting the UPR pathway or inhibiting DNA damage might protect hematopoietic cells of CN patients from leukemogenic transformation, at least to some extent.

GM-CSF treatment is not effective in congenital neutropenia patients due to its inability to activate NAMPT signaling.

Severe congenital neutropenia (CN) is a bone marrow failure syndrome characterized by an absolute neutrophil count (ANC) below 500 cells/µL and recurrent, life-threatening bacterial infections. Treatment with granulocyte colony-stimulating factor (G-CSF) increases the ANC in the majority of CN patients. In contrary, granulocyte-monocyte colony-stimulating factor (GM-CSF) fails to increase neutrophil numbers in CN patients in vitro and in vivo, suggesting specific defects in signaling pathways downstream of GM-CSF receptor. Recently, we detected that G-CSF induces granulopoiesis in CN patients by hyperactivation of nicotinamide phosphoribosyl transferase (NAMPT)/Sirtuin 1 signaling in myeloid cells. Here, we demonstrated that, in contrast to G-CSF, GM-CSF failed to induce NAMPT-dependent granulopoiesis in CN patients. We further identified NAMPT signaling as an essential downstream effector of the GM-CSF pathway in myelopoiesis.

Thrombopoietin induces hematopoiesis from mouse ES cells via HIF-1α-dependent activation of a BMP4 autoregulatory loop.

Understanding the molecular mechanisms underlying hematopoietic differentiation of embryonic stem (ES) cells may help to ascertain the conditions for the in vitro generation of hematopoietic cells. Previously, we found that patients with congenital amegakaryocytic thrombocytopenia (CAMT), who develop pancytopenia early after birth, harbor mutations within the thrombopoietin (TPO) receptor, c-MPL. This knowledge, together with observations in vitro and in vivo, suggests that TPO/c-MPL signaling promotes early hematopoiesis. However, the mechanisms underlying TPO signaling are not fully elucidated. Here, we describe a direct connection between TPO and bone morphogenetic protein 4 (BMP4) signaling pathways in determining the hematopoietic fate of ES cells. Morphogen BMP4 is known to induce early hematopoietic differentiation of ES cells. Treatment of ES cells with TPO induced the autocrine production of BMP4 with concomitant upregulation of the BMP receptor BMPR1A, phosphorylation of SMAD1, 5, 8, and activation of specific BMP4 target genes; this was mediated by TPO-dependent binding of transcription factor HIF-1α to the BMP4 gene promoter. Treatment of ES cells with the BMP antagonist noggin substantially reduced TPO-dependent hematopoietic differentiation of ES cells. Thus, our findings contribute to the establishment of techniques for generating hematopoietic cells from ES cells.

Somatic mosaicism for a NRAS mutation associates with disparate clinical features in RAS-associated leukoproliferative disease: a report of two cases.

RAS-associated leukoproliferative disease (RALD) is a newly classified disease; thus its clinical features and management are not fully understood. The cases of two patients with characteristic features of RALD are described herein. Patient 1 was a 5-month-old female with clinical features typical of autoimmune lymphoproliferative syndrome (ALPS) and markedly elevated TCRαß(+)CD4(-)CD8(-) T cell numbers. Genetic analyses failed to detect an ALPS-related gene mutation; however, whole exome sequencing and other genetic analyses revealed somatic mosaicism for the G13D NRAS mutation. These data were indivative of NRAS-associated RALD with highly elevated αß-double-negative T cells. Patient 2 was a 12-month-old girl with recurrent fever who clearly met the diagnostic criteria for juvenile myelomonocytic leukemia (JMML). Genetic analyses revealed somatic mosaicism, again for the G13D NRAS mutation, suggesting RALD associated with somatic NRAS mosaicism. Notably, unlike most JMML cases, Patient 2 did not require steroids or hematopoietic stem cell transplantation. Genetic analysis of RAS should be performed in patients fulfilling the diagnostic criteria for ALPS in the absence of ALPS-related gene mutations if the patients have elevated αß-double-negative-T cells and in JMML patients if autoimmunity is detected. These clinical and experimental data increase our understanding of RALD, ALPS, and JMML.

Perforation of enteric duplication during chemotherapy for osteosarcoma.

A 9-year-old boy undergoing chemotherapy for conventional osteosarcoma complained of severe abdominal pain associated with rebound tenderness and muscular defense. Abdominal computed tomography indicated intraperitoneal free air. On surgical investigation, a diverticulum-like lesion, perforated at the base, was found on the sidewall of the ileum. The anatomic location of the lesion was indicative of enteric duplication. Although the frequency of complications is very rare, perforations of the digestive tract should be considered in patients suffering severe abdominal pain while receiving chemotherapy.