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Translocations of retinoic acid receptor-α (RARA), typically PML–RARA, are a genetic hallmark of acute promyelocytic leukemia (APL). However, because a small fraction of APL lack translocations of RARA, we focused here on APL cases without RARA translocation to elucidate the molecular etiology of RARA-negative APL. We performed whole-genome sequencing, PCR, and FISH for five APL cases without RARA translocations. Four of five RARA-negative APL cases had translocations involving retinoic acid receptor-β (RARB) translocations, and TBL1XR1–RARB was identified as an in-frame fusion in three cases; one case had an RARB rearrangement detected by FISH, although the partner gene could not be identified. When transduced in cell lines, TBL1XR1–RARB homodimerized and diminished transcriptional activity for the retinoic acid receptor pathway in a dominant-negative manner. TBL1XR1–RARB enhanced the replating capacity of mouse bone marrow cells and inhibited myeloid maturation of human cord blood cells as PML–RARA did. However, the response of APL with RARB translocation to retinoids was attenuated compared with that of PML–RARA, an observation in line with the clinical resistance of RARB-positive APL to ATRA. Our results demonstrate that the majority of RARA-negative APL have RARB translocations, thereby forming a novel, distinct subgroup of APL. TBL1XR1–RARB as an oncogenic protein exerts effects similar to those of PML–RARA, underpinning the importance of retinoic acid pathway alterations in the pathogenesis of APL.Significance: These findings report a novel and distinct genetic subtype of acute promyelocytic leukemia (APL) by illustrating that the majority of APL without RARA translocations harbor RARB translocations. Cancer Res; 78(16); 4452–8. ©2018 AACR.

Supplementary Methods: IRB approval, Genomic analysis, Identification of translocations and determination of their breakpoints, Plasmids Construct, Cell line and reagent, Co-immunoprecipotation and immunoblotting analysis, Luciferase assay, Retrovirus production and transduction, FCM and Morphologic analysis, Human CB cell culture, and Colony replating assay. Supplementary Tables: Table 1. Clinical characteristics of RARA translocation negative APL cases. Table 2. BAC clones for FISH used in this study. Table 3. Primer sequences. Table 4. Somatic mutations observed in RARA-negative APL cases. Supplementary Figures: Figure 1. Morphologic feature and FISH analyisis for RARB translocations. Figure 2. Event-free survival of RARA-negative APL. Figure 3. Determination of TBL1XR1 mutated allele by allele-specific PCR. Figure 4. Constructs of TBL1XR1-RARB and other RARA fusions. Figure 5. Morphologic change in U937. Figure 6. Colony replating assay with mouse bone marrow. Figure 7. Colony replating assay with human cord blood.

The chimeric oncogene AML1-MTG8 (RUNX1-RUNX1T1) is generated in t(8;21) acute myeloid leukemia (AML). Here, we report a novel interaction of MTG8/RUNX1T1/ETO with UBC9/UBE2I. AML1-MTG8 protein also interacted with UBC9, suggesting a role in leukemogenesis. Overexpression of UBC9 in Kasumi-1 attenuated myeloid differentiation induced by all-trans retinoic acid, G-CSF, and GM-CSF (AGGM), which was judged by suppression of CD11b. In addition, the UBC9 inhibitor 2-D08 accelerated myeloid differentiation induced by AGGM in two t(8;21) AML cell lines, Kasumi-1 and SKNO-1. These data suggest that UBC9 may play a role in leukemogenesis in t(8;21) AML by working with AML1-MTG8 to suppress myeloid differentiation. Therefore, UBC9 may be a good target for new differentiation therapy against t(8;21) AML.

Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in de novo guanine nucleotide synthesis pathway. Although IMPDH inhibitors are widely used as effective immunosuppressants, their antitumor effects have not been proven in the clinical setting. Here, we found that acute myeloid leukemias (AMLs) with MLL-fusions are susceptible to IMPDH inhibitors in vitro. We also showed that alternate-day administration of IMPDH inhibitors suppressed the development of MLL-AF9-driven AML in vivo without having a devastating effect on immune function. Mechanistically, IMPDH inhibition induced overactivation of Toll-like receptor (TLR)-TRAF6-NF-κB signaling and upregulation of an adhesion molecule VCAM1, which contribute to the antileukemia effect of IMPDH inhibitors. Consequently, combined treatment with IMPDH inhibitors and the TLR1/2 agonist effectively inhibited the development of MLL-fusion AML. These findings provide a rational basis for clinical testing of IMPDH inhibitors against MLL-fusion AMLs and potentially other aggressive tumors with active TLR signaling.

SLC29A3, also known as ENT3, is a lysosomal transmembrane protein that transports nucleosides from the lysosomes to the cytoplasm1. Loss-of-function mutations inSLC29A3cause lysosomal nucleoside storage and histiocytosis: phagocyte accumulation in multiple organs2,3. However, little is known about the mechanism through which lysosomal nucleoside storage drives histiocytosis. Herein, histiocytosis inSlc29a3−/−mice was demonstrated to depend on TLR7, which senses a combination of nucleosides and oligoribonucleotides4,5. TLR7 responded to lysosomal nucleoside storage and enhanced proliferation of Ly6ChiCX3CR1lowimmature monocytes and their maturation into Ly6Clowphagocytes inSlc29a3−/−mice. Because accumulated nucleosides primarily originated from cell corpse phagocytosis, TLR7 in immature monocytes recognized nucleoside storage as lysosomal stress and increased phagocyte numbers. This non-inflammatory compensatory response is referred to as the TLR7 stress response where Syk, GSK3β, β-catenin, and mTORC1 serve as downstream signalling molecules. In SLC29A3 disorders, histiocytosis accompanies inflammation6,7. Nucleoside storage failed to induce pro-inflammatory cytokine production inSlc29a3−/−mice, but enhanced ssRNA-dependent pro-inflammatory cytokine production in Ly6Chiclassical monocytes and peripheral macrophages, not proliferating immature monocytes. Patient-derived monocytes harbouring G208RSLC29A3mutation showed higher survival and proliferation in the presence of M-CSF and produced larger amounts of IL-6 upon ssRNA stimulation than did those derived from healthy subjects. A TLR8 antagonist inhibited the survival/proliferation of patient-derived macrophages. These results demonstrated that TLR7/8 responses to lysosomal nucleoside stress drive SLC29A3 disorders.

Immunotherapy has attracted considerable attention as a therapeutic strategy for cancers including acute myeloid leukemia (AML). In this study, we found that the development of several aggressive subtypes of AML was retarded in Rag2-/- mice despite the lack of B and T lymphocytes, even compared to the immunologically normal C57BL/6 mice. Furthermore, an orally active p53-activating drug showed stronger antileukemia effect on AML in Rag2-/- mice than C57BL/6 mice. Intriguingly, NK cells in Rag2-/- mice were increased in number, highly expressed activation markers, and showed increased cytotoxicity to leukemia cells in a coculture assay. B2m depletion that triggers missing-self recognition of NK cells impaired the growth of AML cells in vivo. In contrast, NK cell depletion accelerated AML progression in Rag2-/- mice. We also found that immunogenicity of AML kept changing during tumor evolution, showing a trend that AMLs with strong leukemogenicity were susceptible to NK cell-mediated tumor suppression in Rag2-/- mice. Thus, our study highlighted the critical role of NK cells in suppressing the development of certain subtypes of AML, and demonstrated that Rag2-/- mice, which are generally considered as “immunodeficient” due to the lack of functional lymphocytes, in fact have hyperactive NK cells with the enhanced antileukemia immunity.

Hematopoietic stem cells (HSCs) undergo progressive functional decline over time due to both internal and external stressors, leading to aging of the hematopoietic system. A comprehensive understanding of the molecular mechanisms underlying HSC aging will be valuable in developing novel therapies for HSC rejuvenation and to prevent the onset of several age-associated diseases and hematological malignancies. This review considers the general causes of HSC aging that range from cell-intrinsic factors to cell-extrinsic factors. In particular, epigenetics and inflammation have been implicated in the linkage of HSC aging, clonality, and oncogenesis. The challenges in clarifying mechanisms of HSC aging have accelerated the development of therapeutic interventions to rejuvenate HSCs, the major goal of aging research; these details are also discussed in this review.

Clonal hematopoiesis of indeterminate potential (CHIP) is an age-associated phenomenon characterized by clonal expansion of blood cells harboring somatic mutations in hematopoietic genes, including DNMT3A, TET2, and ASXL1. Clinical evidence suggests that CHIP is highly prevalent and associated with poor prognosis in solid-tumor patients. However, whether blood cells with CHIP mutations play a causal role in promoting the development of solid tumors remained unclear. Using conditional knock-in mice that express CHIP-associated mutant Asxl1 (Asxl1-MT), we showed that expression of Asxl1-MT in T cells, but not in myeloid cells, promoted solid-tumor progression in syngeneic transplantation models. We also demonstrated that Asxl1-MT-expressing blood cells accelerated the development of spontaneous mammary tumors induced by MMTV-PyMT. Intratumor analysis of the mammary tumors revealed the reduced T-cell infiltration at tumor sites and programmed death receptor-1 (PD-1) upregulation in CD8

In classical Hodgkin lymphoma (cHL)—characterized by the presence of Hodgkin and Reed-Sternberg (HRS) cells—tumor-associated macrophages (TAMs) play a pivotal role in tumor formation. However, the significance of direct contact between HRS cells and TAMs has not been elucidated. HRS cells and TAMs are known to express PD-L1, which leads to PD-1+ CD4+ T cell exhaustion in cHL. Here, we found that PD-L1/L2 expression was elevated in monocytes co-cultured with HRS cells within 1 h, but not in monocytes cultured with supernatants of HRS cells. Immunofluorescence analysis of PD-L1/L2 revealed that their upregulation resulted in membrane transfer called “trogocytosis” from HRS cells to monocytes. PD-L1/L2 upregulation was not observed in monocytes co-cultured with PD-L1/L2-deficient HRS cells, validating the hypothesis that there is a direct transfer of PD-L1/L2 from HRS cells to monocytes. In the patients, both ligands (PD-L1/L2) were upregulated in TAMs in contact with HRS cells, but not in TAMs distant from HRS cells, suggesting that trogocytosis occurs in cHL patients. Taken together, trogocytosis may be one of the mechanisms that induces rapid upregulation of PD-L1/L2 in monocytes to evade antitumor immunity through the suppression of T cells as mediated by MHC antigen presentation.

The negative regulator of p53, MDM2, is frequently overexpressed in acute myeloid leukemia (AML) that retains wild-type TP53 alleles. Targeting of p53-MDM2 interaction to reactivate p53 function is therefore an attractive therapeutic approach for AML. Here we show that an orally active inhibitor of p53-MDM2 interaction, DS-5272, causes dramatic tumor regressions of MLL-AF9-driven AML in vivo with a tolerable toxicity. However, the antileukemia effect of DS-5272 is markedly attenuated in immunodeficient mice, indicating the critical impact of systemic immune responses that drive p53-mediated leukemia suppression. In relation to this, DS-5272 triggers immune-inflammatory responses in MLL-AF9 cells including upregulation of Hif1α and PD-L1, and inhibition of the Hif1α-PD-L1 axis sensitizes AML cells to p53 activation. We also found that NK cells are important mediators of antileukemia immunity. Our study showed the potent activity of a p53-activating drug against AML, which is further augmented by antitumor immunity., MDM2 is frequently overexpressed in acute myeloid leukaemia leading to p53 inactivation. Here, the authors are demonstrating that an inhibitor of p53-MDM2 interaction, DS-5272, induce in vivo tumour regression through immune response regulation.

This study aimed to evaluate the sequential changes in the proportion of anemia among young women over eight years after the Great East Japan Earthquake in 2011 using a prospective study of the Fukushima Health Management Survey. This study focused on the women aged between 20 and 44 who lived in the evacuation area of the nuclear power plant accident. The yearly age-adjusted proportion of anemia was accessed with data between July 2011 and March 2019. A total of 9,198 women participated in the health checkup in 2011, albeit the participation was decreased to 1,241 in 2018. The age-adjusted proportion of anemia was 16.7% in 2012 and then declined after 2013 (p with Cochran-Armitage trend test = 0.03). The multivariate regression analysis identified 2 of body mass index (BMI), no history of smoking, and no habitual alcohol use as independent baseline characteristics predictive of temporality anemic condition after the disaster (Adjusted odds ratios [95% confidence interval]; 1.98 [1.43–2.74], 1.85 [1.21–2.83], and 1.42 [1.07–1.90], respectively). Thus, women with low BMI and healthier habits might risk temporarily anemic status after the disaster. Our findings signal the importance of preventing anemia in young women after the disaster.

Numerous strategies exist to isolate hematopoietic stem cells (HSCs) using complex combinations of markers and flow cytometry. However, robust identification of HSCs using imaging techniques is substantially more challenging which has prompted the recent development of HSC reporter mice. To date, none of the molecules used in these reporters have been useful for human HSC identification. Here we report that PLXDC2 is a useful marker for both mouse and human HSCs. Using a green fluorescent protein (GFP) knock-in at the Plxdc2 locus in mice (hereafter denoted as Plxdc2-GFP), we showed that Plxdc2-GFP is highly expressed in HSCs with 1 in 2.8 Plxdc2-GFP+CD150+ cells giving long-term multi-lineage reconstitution in transplantation. Moreover, we developed a novel human PLXDC2 antibody and showed that human PLXDC2+ HSCs have stronger long-term multilineage reconstitution ability compared with PLXDC2- HSCs in a xenograft model. Thus, our study identifies PLXDC2 as a highly relevant molecule in HSC identification, potentially allowing greater purity and live in vivo tracking of these cells.SummaryTo date, few molecules are available for isolation of HSCs across species. The present study shows that PLXDC2 is a highly useful molecule for isolation of HSCs, which works across mouse and human.

Clonal hematopoiesis (CH) harboring a leukemia-related mutation has been recently found in about 10% of healthy elderly individuals, which has been attracting attention. Although most people with CH do not develop hematological malignancies, some may develop hematological malignancies 10 times more frequently than age-matched controls. On the other hand, compared to age-matched controls, the probability of developing cardiovascular diseases in people with CH is 2-fold, which is thought to shorten the life expectancy. Moreover, one out of four patients with solid cancer and one out of two patients with aplastic anemia, whose mutation profiles overlap with but are distinct from common mutations identified with CH of elderly people, harbor CH. The study of CH has just begun, and there are many unknowns. In an aging society of unprecedented proportions, which is also attracting attention from the society, the establishment of a new research field that investigates CH in the near future is likely.

Leukemia stem cells (LSCs) in chronic myeloid leukemia (CML) are quiescent, insensitive to BCR-ABL1 tyrosine kinase inhibitors (TKIs) and responsible for CML relapse. Therefore, eradicating quiescent CML LSCs is a major goal in CML therapy. Here, using a novel G0 marker (G0M), we narrowed down CML LSCs as G0M- and CD27- double positive cells among the conventional CML LSCs. Whole transcriptome analysis revealed NF-κB activation via inflammatory signals in imatinib-insensitive quiescent CML LSCs. Blocking NF-κB signals by IRAK1/4 inhibitors together with imatinib eradicated mouse and human CML LSCs. Intriguingly, IRAK1/4 inhibitors attenuated PD-L1 expression on CML LSCs, and blocking PD-L1 together with imatinib also effectively eradicated CML LSCs in the presence of T cell immunity. Thus, IRAK1/4 inhibitors could eradicate CML LSCs through inhibiting NF-ΚB activity and reducing PD-L1 expression. Collectively, the combination of TKIs and IRAK1/4 inhibitors is an attractive strategy to achieve a radical cure of CML.

Kin of irregular chiasm-like 3 (Kirrel3), a member of the immunoglobulin superfamily, is expressed in the central nervous system during development and in adulthood. It has been reported that Kirrel3 is involved in the axonal fasciculation in the olfactory bulb, the neuronal migration in the pontine nucleus, and the synapse formation in the hippocampal neurons in mice. Although KIRREL3 mutations have been implicated in autism spectrum disorder and intellectual disability in humans, the comprehensive expression pattern of Kirrel3 in the adult brain is not fully understood. To better visualize Kirrel3 expression pattern and to gain insight into the role of Kirrel3 in the brain, we investigated the expression of Kirrel3 in the adult brain of Kirrel3-heterozygous (Kirrel3+/-) mice, in which Kirrel3-expressing cells could be identified by the expression of β-galactosidase (β-gal) in the nucleus of cells. The strong expression of β-gal was observed in the hippocampus, cerebral cortex, olfactory bulb, amygdala, thalamus, and cerebellum. In the hippocampus, β-gal was detected in the dentate gyrus and in the ventral parts of CA1 and CA3, which are known to be involved in the social recognition memory. Within the cerebral cortex, many cells with β-gal expression were observed in the olfactory area and auditory area. In the striatum, neurons with β-gal expression were mainly observed in the ventral striatum. Expression of β-gal was observed in all layers in the cerebellum and olfactory bulb, except for the olfactory nerve layer. Double-immunofluorescence staining of β-galactosidase with neuronal markers revealed that β-gal expression was exclusively detected in neurons. These results suggest that Kirrel3 may be involved in the maintenance of neuronal networks, such as the maintenance of synaptic connectivity and plasticity in the motor, sensory, and cognitive circuits of adult brain.

Recent high-throughput genome-wide sequencing studies have identified recurrent somatic mutations in myeloid neoplasms. An epigenetic regulator, Additional sex combs-like 1 (ASXL1), is one of the most frequently mutated genes in all subtypes of myeloid malignancies. ASXL1 mutations are also frequently detected in clonal hematopoiesis, which is associated with an increased risk of mortality. Therefore, it is important to understand how ASXL1 mutations contribute to clonal expansion and myeloid transformation in hematopoietic cells. Studies using ASXL1-depleted human hematopoietic cells and Asxl1 knockout mice have shown that deletion of wild-type ASXL1 protein leads to impaired hematopoiesis and accelerates myeloid malignancies via loss of interaction with polycomb repressive complex 2 proteins. On the other hand, ASXL1 mutations in myeloid neoplasms typically occur near the last exon and result in the expression of C-terminally truncated mutant ASXL1 protein. Biological studies and biochemical analyses of this variant have shed light on its dominant-negative and gain-of-function features in myeloid transformation via a variety of epigenetic changes. Based on these results, it would be possible to establish novel promising therapeutic strategies for myeloid malignancies harboring ASXL1 mutations by blocking interactions between ASXL1 and associating epigenetic regulators. Here, we summarize the clinical implications of ASXL1 mutations, the role of wild-type ASXL1 in normal hematopoiesis, and oncogenic functions of mutant ASXL1 in myeloid neoplasms.

Myelodysplastic syndrome (MDS) is a clonal disorder of hematopoietic stem cells (HSCs), characterized by ineffective hematopoiesis and frequent progression to leukemia. It has long remained unresolved how MDS cells, which are less proliferative, inhibit normal hematopoiesis and eventually dominate the bone marrow space. Despite several studies implicating mesenchymal stromal or stem cells (MSCs), a principal component of the HSC niche, in the inhibition of normal hematopoiesis, the molecular mechanisms underlying this process remain unclear. Here, we demonstrate that both human and mouse MDS cells perturb bone metabolism by suppressing the osteolineage differentiation of MSCs, which impairs the ability of MSCs to support normal HSCs. Enforced MSC differentiation rescues the suppressed normal hematopoiesis in both in vivo and in vitro MDS models. Intriguingly, the suppression effect is reversible and mediated by extracellular vesicles (EVs) derived from MDS cells. These findings shed light on the novel MDS EV-MSC axis in ineffective hematopoiesis.

RUNX1 is a member of RUNX transcription factors and plays important roles in hematopoiesis. RUNX1 function is under the tight control through posttranslational modifications, including phosphorylation and ubiquitination. We previously developed a luminescence-based binding assay (AlphaScreen) to systematically detect RUNX1-interacting E3 ubiquitin ligases. In this study, we showed that a nuclear ubiquitin ligase RNF38 induced ubiquitination of RUNX1. RNF38-induced RUNX1 ubiquitination did not promote RUNX1 degradation, but rather stabilized RUNX1 protein. We also found that RNF38 enhanced RUNX1-mediated transcriptional repression of the erythroid master regulator KLF1 in K562 cells. Consequently, RNF38 cooperated with RUNX1 to inhibit erythroid differentiation of K562 cells. Thus, our study identified RNF38 as a novel E3 ligase that modifies RUNX1 function without inducing its degradation.

Somatic mutations of ASXL1 are frequently detected in age-related clonal hematopoiesis (CH). However, how ASXL1 mutations drive CH remains elusive. Using knockin (KI) mice expressing a C-terminally truncated form of ASXL1-mutant (ASXL1-MT), we examined the influence of ASXL1-MT on physiological aging in hematopoietic stem cells (HSCs). HSCs expressing ASXL1-MT display competitive disadvantage after transplantation. Nevertheless, in genetic mosaic mouse model, they acquire clonal advantage during aging, recapitulating CH in humans. Mechanistically, ASXL1-MT cooperates with BAP1 to deubiquitinate and activate AKT. Overactive Akt/mTOR signaling induced by ASXL1-MT results in aberrant proliferation and dysfunction of HSCs associated with age-related accumulation of DNA damage. Treatment with an mTOR inhibitor rapamycin ameliorates aberrant expansion of the HSC compartment as well as dysregulated hematopoiesis in aged ASXL1-MT KI mice. Our findings suggest that ASXL1-MT provokes dysfunction of HSCs, whereas it confers clonal advantage on HSCs over time, leading to the development of CH., ASXL1 mutations are frequently found in age-related clonal haemaotopoiesis (CH), but how they drive CH is unclear. Here the authors show that expression of C-terminal truncated ASXL1 in haematopoietic stem cells (HSCs) leads to Akt de-ubiquitination, activated Akt/mTOR signaling, and aberrant HSC proliferation.

Cellular behavior is governed by the complex gene regulatory networks. Although studies have revealed diverse roles of individual genes, it has been a challenge to record or control the sequential genetic events in living cells. In this study, we designed two cellular chain reaction systems that enable sequential sgRNA expression in mammalian cells using a nickase Cas9 tethering of a cytosine nucleotide deaminase (nCas9-CDA). In these systems, the thymidine (T)-to-cytosine (C) substitutions in the scaffold region of sgRNA or TATA box containing loxP sequence (TATAloxP) are corrected by the nCas9-CDA, which leads to expression of next sgRNA. These reactions can proceed several times, thus generating cellular chain reactions. As a proof of the concept, we established a chain reaction through the repair of sgRNA scaffold mutations in 293T cells. Importantly, the results obtained in yeast or in vitro were not consistent with those in mammalian cells, suggesting that the in vivo chain reactions need to be optimized in appropriate cellular contexts. Our system may lay the foundation for building cellular chain reaction systems that have a broad utility in the future biomedical research.

Tomato yellow leaf curl virus (TYLCV) and tomato chlorosis virus (ToCV) were transmitted by the sweet potato whitefly Bemisia tabaci (Gennadius) and cause serious yield losses on tomato around the world. To understand the actual situation of co-infection of TYLCV and ToCV of individual whiteflies, we developed multiplex RT-PCR combined with co-extraction of DNA and RNA from a single whitefly. First, a nucleic acid extraction method previously reported was modified and adopted to obtain the RNA-DNA mixture including TYLCV and ToCV in a simple form without manual homogenization. Second, primers were newly designed in actin gene of B. tabaci for the confirmation of extraction and PCR success, and multiplex RT-PCR method was developed using specific primer sets for TYLCV, ToCV and B. tabaci. This method enables the detection of TYLCV and ToCV from a single insect and efficient use of field samples obtained using sticky traps. The method will be useful to monitor infection status of TYLCV and ToCV in the field while reducing labor and cost.

Summary: Paraspeckles are membraneless organelles formed through liquid-liquid phase separation and consist of multiple proteins and RNAs, including NONO, SFPQ, and NEAT1. The role of paraspeckles and the component NONO in hematopoiesis remains unknown. In this study, we show histone modifier ASXL1 is involved in paraspeckle formation. ASXL1 forms phase-separated droplets, upregulates NEAT1 expression, and increases NONO-NEAT1 interactions through the C-terminal intrinsically disordered region (IDR). In contrast, a pathogenic ASXL mutant (ASXL1-MT) lacking IDR does not support the interaction of paraspeckle components. Furthermore, paraspeckles are disrupted and Nono localization is abnormal in the cytoplasm of hematopoietic stem and progenitor cells (HSPCs) derived from ASXL1-MT knockin mice. Nono depletion and the forced expression of cytoplasmic NONO impair the repopulating potential of HSPCs, as does ASXL1-MT. Our study indicates a link between ASXL1 and paraspeckle components in the maintenance of normal hematopoiesis.

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder caused by constitutively active BCR-ABL1 tyrosine kinase resulting from the t(9;22) Philadelphia translocation. Imatinib, a BCR-ABL1 tyrosine kinase inhibitor (TKI), is a revolutionary molecular target inhibitor for CML. However, leukemic stem cells (LSCs) eventually become resistant to imatinib and thereby cause relapse. The next-generation BCR-ABL1 TKI dasatinib is also unable to eliminate CML LSCs. On the other hand, the third-generation BCR-ABL1 TKI ponatinib is not well studied in terms of its efficacy on CML LSCs. Here, we evaluate the efficacy of ponatinib against CML LSC-containing lin–Sca-1+c-Kit+ (LSK) cells using a mouse CML-like model. To this end, we compared the efficacy of imatinib, dasatinib, and ponatinib on CML LSK cells and showed that ponatinib is more effective at eliminating CML LSK cells. Our results suggest that ponatinib could be potentially useful for achieving treatment-free remission in CML patients.

Inoue et al. show that a small population of germinal center–derived CD38intBcl6hi/intEfnb1+ cells with lower mTORC1 activity favors a memory B cell fate. This population also has higher levels of Bcl2 and surface BCR that, in turn, contributes to their survival and development., A still unanswered question is what drives the small fraction of activated germinal center (GC) B cells to become long-lived quiescent memory B cells. We found here that a small population of GC-derived CD38intBcl6hi/intEfnb1+ cells with lower mTORC1 activity favored the memory B cell fate. Constitutively high mTORC1 activity led to defects in formation of the CD38intBcl6hi/intEfnb1+ cells; conversely, decreasing mTORC1 activity resulted in relative enrichment of this memory-prone population over the recycling-prone one. Furthermore, the CD38intBcl6hi/intEfnb1+ cells had higher levels of Bcl2 and surface BCR that, in turn, contributed to their survival and development. We also found that downregulation of Bcl6 resulted in increased expression of both Bcl2 and BCR. Given the positive correlation between the strength of T cell help and mTORC1 activity, our data suggest a model in which weak help from T cells together with provision of an increased survival signal are key for GC B cells to adopt a memory B cell fate.

Quiescent hematopoietic stem cells (HSCs) are typically dormant, and only a few quiescent HSCs are active. The relationship between “dormant” and “active” HSCs remains unresolved. Here we generated a G0 marker (G0M) mouse line that visualizes quiescent cells. G0M identifies a small population of active HSCs (G0Mlow), which are distinct from dormant HSCs (G0Mhigh), within the conventional quiescent HSC fraction. Single-cell RNA-Seq analyses showed that the gene expression profiles of these populations were nearly identical, yet differed in their Cdk4/6 activity. Furthermore high-throughput small molecule screening revealed that high concentrations of cytoplasmic calcium ([Ca2+]c) were linked to dormancy of HSCs. These findings indicate that G0M separates dormant and active adult HSCs, which are regulated by Cdk4/6 and [Ca2+]c. This G0M mouse line represents a useful resource for investigating physiologically important stem cell subpopulations., Wellcome Trust, Takeda Science Foundation, The Tokyo Biochemical Research Foundation