Application of prime editing system to introduce TP53 R248Q hotspot mutation in acute lymphoblastic leukemia cell line.

In childhood acute lymphoblastic leukemia (ALL), TP53 gene mutation is associated with chemoresistance in a certain population of relapsed cases. To directly verify the association of TP53 gene mutation with chemoresistance of relapsed childhood ALL cases and improve their prognosis, the development of appropriate human leukemia models having TP53 mutation in the intrinsic gene is required. Here, we sought to introduce R248Q hotspot mutation into the intrinsic TP53 gene in an ALL cell line, 697, by applying a prime editing (PE) system, which is a versatile genome editing technology. The PE2 system uses an artificial fusion of nickase Cas9 and reverse-transcriptase to directly place new genetic information into a target site through a reverse transcriptase template in the prime editing guide RNA (pegRNA). Moreover, in the advanced PE3b system, single guide RNA (sgRNA) matching the edited sequence is also introduced to improve editing efficiency. The initially obtained MDM2 inhibitor-resistant PE3b-transfected subline revealed disrupted p53 transactivation activity, reduced p53 target gene expression, and acquired resistance to chemotherapeutic agents and irradiation. Although the majority of the subline acquired the designed R248Q and adjacent silent mutations, the insertion of the palindromic sequence in the scaffold hairpin structure of pegRNA and the overlap of the original genomic DNA sequence were frequently observed. Targeted next-generation sequencing reconfirmed frequent edit errors in both PE2 and PE3b-transfected 697 cells, and it revealed frequent successful edits in HEK293T cells. These observations suggest a requirement for further modification of the PE2 and PE3b systems for accurate editing in leukemic cells.

A novel anticancer quinolone, (R)-WAC-224, has anti-leukemia activities against acute myeloid leukemia.

Approximately 60%-80% of patients who achieve complete remission eventually relapse after conventional chemotherapy and have poor prognoses despite the recent advances of novel anticancer agents. Continuing development of more effective novel treatments for acute myeloid leukemia (AML) is necessary. We developed (R)-WAC-224 (R-WAC), which is an anticancer quinolone, targeting topoisomerase II. This study evaluated the anti-leukemia potential of R-WAC or racemic WAC-224 (WAC) in vitro and in vivo. R-WAC significantly inhibited the human AML cell line proliferation (MV4-11, HL60, and KG1a), which was comparable to daunorubicin and cytarabine, not affected by P-glycoprotein overexpression. WAC did neither increase serum troponin-T nor decrease the crypt numbers in the small intestine, indicating WAC was less toxic than doxorubicin. R-WAC monotherapy demonstrated prolonged survival in the AML mice model and inhibited tumor growth in the MV4-11 xenograft mice model. Moreover, the combination of R-WAC and cytarabine demonstrated more active anti-leukemia effects than daunorubicin and cytarabine. Finally, R-WAC inhibited the colony-forming abilities using primary AML cells. These results indicate that R-WAC is a promising therapeutic agent for AML.

Tumour-derived exosomes promote the induction of monocytic myeloid-derived suppressor cells from peripheral blood mononuclear cells by delivering miR-106a-5p and miR-146a-5p in multiple myeloma.

The shift of the tumour immune microenvironment to a suppressive state promotes not only the development and progression of the disease in multiple myeloma (MM) but also the development of resistance to immunotherapy. We previously demonstrated that myeloma cells can induce monocytic myeloid-derived suppressor cells (M-MDSCs) from healthy peripheral blood mononuclear cells (PBMCs) via the concomitant secretion of CC motif chemokine ligand 5 (CCL5) and macrophage migration inhibitory factor (MIF), but an unknown mediator also promotes M-MDSC induction. This study demonstrates that miR-106a-5p and miR-146a-5p delivered by tumour-derived exosomes (TEXs) from myeloma cells play essential roles in M-MDSC induction in MM. MiR-106a-5p and miR-146a-5p upregulate various immunosuppressive/inflammatory molecules in PBMCs, such as IDO1, CD38, programmed death-ligand 1, CCL5 or MYD88, which are involved in interferon (IFN)-α response, IFN-γ response, inflammatory response, tumour necrosis factor-α signalling and Interleukin-6-JAK-STAT3 signalling. These molecular features mirror the increases in myeloid cellular compartments of PBMCs when co-cultured with myeloma cells. MiR-106a-5p and miR-146a-5p have a compensatory relationship, and these two miRNAs collaborate with CCL5 and MIF to promote M-MDSC induction. Collectively, novel therapeutic candidates may be involved in TEX-mediated sequential cellular and molecular events underlying M-MDSC induction, potentially improving the efficacy of immunotherapy.

MOZ/ENL complex is a recruiting factor of leukemic AF10 fusion proteins.

Changes in the transcriptional machinery cause aberrant self-renewal of non-stem hematopoietic progenitors. AF10 fusions, such as CALM-AF10, are generated via chromosomal translocations, causing malignant leukemia. In this study, we demonstrate that AF10 fusion proteins cause aberrant self-renewal via ENL, which binds to MOZ/MORF lysine acetyltransferases (KATs). The interaction of ENL with MOZ, via its YEATS domain, is critical for CALM-AF10-mediated leukemic transformation. The MOZ/ENL complex recruits DOT1L/AF10 fusion complexes and maintains their chromatin retention via KAT activity. Therefore, inhibitors of MOZ/MORF KATs directly suppress the functions of AF10 fusion proteins, thereby exhibiting strong antitumor effects on AF10 translocation-induced leukemia. Combinatorial inhibition of MOZ/MORF and DOT1L cooperatively induces differentiation of CALM-AF10-leukemia cells. These results reveal roles for the MOZ/ENL complex as an essential recruiting factor of the AF10 fusion/DOT1L complex, providing a rationale for using MOZ/MORF KAT inhibitors in AF10 translocation-induced leukemia.

Ribosome profiling analysis reveals the roles of DDX41 in translational regulation.

DDX41 mutation has been observed in myeloid malignancies including myelodysplastic syndromes and acute myeloid leukemia, but the underlying causative mechanisms of these diseases have not been fully elucidated. The DDX41 protein is an ATP-dependent RNA helicase with roles in RNA metabolism. We previously showed that DDX41 is involved in ribosome biogenesis by promoting the processing of newly transcribed pre-ribosomal RNA. To build on this finding, in this study, we leveraged ribosome profiling technology to investigate the involvement of DDX41 in translation. We found that DDX41 knockdown resulted in both translationally increased and decreased transcripts. Both gene set enrichment analysis and gene ontology analysis indicated that ribosome-associated genes were translationally promoted after DDX41 knockdown, in part because these transcripts had significantly shorter transcript length and higher transcriptional and translational levels. In addition, we found that transcripts with 5'-terminal oligopyrimidine motifs tended to be translationally upregulated when the DDX41 level was low. Our data suggest that a translationally regulated feedback mechanism involving DDX41 may exist for ribosome biogenesis.

[Mouse models of hematological diseases using genome editing technology].

The impact of gene-editing technology has rapidly expanded into developmental engineering. Using this technology, gene targeting in mice can be performed within 2-3 months, which is a much shorter timespan than that required while using embryonic stem cell-based conventional methods, which require nearly two years. In addition, genome-editing technology omits several skillful laborious steps. This review describes the prominent merits of gene targeting using this recently established and still ongoing technology in the field of hematology. In addition, the experience of the authors is reviewed to identify and characterize genes involved in the loss of the long arm of chromosome 7 in myeloid malignancies and highlight the significance of establishing the mouse model of human diseases.

DDX41 coordinates RNA splicing and transcriptional elongation to prevent DNA replication stress in hematopoietic cells.

Myeloid malignancies with DDX41 mutations are often associated with bone marrow failure and cytopenia before overt disease manifestation. However, the mechanisms underlying these specific conditions remain elusive. Here, we demonstrate that loss of DDX41 function impairs efficient RNA splicing, resulting in DNA replication stress with excess R-loop formation. Mechanistically, DDX41 binds to the 5' splice site (5'SS) of coding RNA and coordinates RNA splicing and transcriptional elongation; loss of DDX41 prevents splicing-coupled transient pausing of RNA polymerase II at 5'SS, causing aberrant R-loop formation and transcription-replication collisions. Although the degree of DNA replication stress acquired in S phase is small, cells undergo mitosis with under-replicated DNA being remained, resulting in micronuclei formation and significant DNA damage, thus leading to impaired cell proliferation and genomic instability. These processes may be responsible for disease phenotypes associated with DDX41 mutations.

Multi-omics analysis defines highly refractory RAS burdened immature subgroup of infant acute lymphoblastic leukemia.

KMT2A-rearranged infant acute lymphoblastic leukemia (ALL) represents the most refractory type of childhood leukemia. To uncover the molecular heterogeneity of this disease, we perform RNA sequencing, methylation array analysis, whole exome and targeted deep sequencing on 84 infants with KMT2A-rearranged leukemia. Our multi-omics clustering followed by single-sample and single-cell inference of hematopoietic differentiation establishes five robust integrative clusters (ICs) with different master transcription factors, fusion partners and corresponding stages of B-lymphopoietic and early hemato-endothelial development: IRX-type differentiated (IC1), IRX-type undifferentiated (IC2), HOXA-type MLLT1 (IC3), HOXA-type MLLT3 (IC4), and HOXA-type AFF1 (IC5). Importantly, our deep mutational analysis reveals that the number of RAS pathway mutations predicts prognosis and that the most refractory subgroup of IC2 possesses 100% frequency and the heaviest burden of RAS pathway mutations. Our findings highlight the previously under-appreciated intra- and inter-patient heterogeneity of KMT2A-rearranged infant ALL and provide a rationale for the future development of genomics-guided risk stratification and individualized therapy.

Aberrant EVI1 splicing contributes to EVI1-rearranged leukemia.

Detailed genomic and epigenomic analyses of MECOM (the MDS1 and EVI1 complex locus) have revealed that inversion or translocation of chromosome 3 drives inv(3)/t(3;3) myeloid leukemias via structural rearrangement of an enhancer that upregulates transcription of EVI1. Here, we identify a novel, previously unannotated oncogenic RNA-splicing derived isoform of EVI1 that is frequently present in inv(3)/t(3;3) acute myeloid leukemia (AML) and directly contributes to leukemic transformation. This EVI1 isoform is generated by oncogenic mutations in the core RNA splicing factor SF3B1, which is mutated in >30% of inv(3)/t(3;3) myeloid neoplasm patients and thereby represents the single most commonly cooccurring genomic alteration in inv(3)/t(3;3) patients. SF3B1 mutations are statistically uniquely enriched in inv(3)/t(3;3) myeloid neoplasm patients and patient-derived cell lines compared with other forms of AML and promote mis-splicing of EVI1 generating an in-frame insertion of 6 amino acids at the 3' end of the second zinc finger domain of EVI1. Expression of this EVI1 splice variant enhanced the self-renewal of hematopoietic stem cells, and introduction of mutant SF3B1 in mice bearing the humanized inv(3)(q21q26) allele resulted in generation of this novel EVI1 isoform in mice and hastened leukemogenesis in vivo. The mutant SF3B1 spliceosome depends upon an exonic splicing enhancer within EVI1 exon 13 to promote usage of a cryptic branch point and aberrant 3' splice site within intron 12 resulting in the generation of this isoform. These data provide a mechanistic basis for the frequent cooccurrence of SF3B1 mutations as well as new insights into the pathogenesis of myeloid leukemias harboring inv(3)/t(3;3).

Design, synthesis and biological evaluation of 2-pyrrolone derivatives as radioprotectors.

It is known that p53 is an important transcription factor and plays a central role in ionizing radiation (IR)-induced DNA damage responses such as cell cycle arrest, DNA repair and apoptosis. We previously reported that regulating p53 protein is an effective strategy for modulating cell fate by reducing the acute side effects of radiation therapy. Herein, we report on the discovery of STK160830 as a new radioprotector from a chemical library at The University of Tokyo and the design, synthesis and biological evaluation of its derivatives. The radioprotective activity of STK160830 itself and its derivatives that were synthesized in this work was evaluated using a leukemia cell line, MOLT-4 cells as a model of normal cells that express the p53 protein in a structure-activity relationships (SAR) study. The experimental results suggest that a direct relationship exists between the inhibitory effect of these STK160830 derivatives on the expression level of p53 and their radioprotective activity and that the suppression of p53 by STK160830 derivatives contribute to protecting MOLT-4 cells from apoptosis that is induced by exposure to radiation.

Glucocorticoid receptor gene mutations confer glucocorticoid resistance in B-cell precursor acute lymphoblastic leukemia.

Glucocorticoid (GC) is a key drug in the treatment of B-cell precursor acute lymphoblastic leukemia (BCP-ALL), and the initial GC response is an important prognostic factor. GC receptors play an essential role in GC sensitivity, and somatic mutations of the GC receptor gene, NR3C1, are reportedly identified in some BCP-ALL cases, particularly at relapse. Moreover, associations of somatic mutations of the CREB-binding protein (CREBBP) and Wolf-Hirschhorn syndrome candidate 1 (WHSC1) genes with the GC-resistance of ALL have been suggested. However, the significance of these mutations in the GC sensitivity of BCP-ALL remains to be clarified in the intrinsic genes. In the present study, we sequenced NR3C1, WHSC1, and CREBBP genes in 99 BCP-ALL and 22 T-ALL cell lines (32 and 67 cell lines were known to be established at diagnosis and at relapse, respectively), and detected their mutations in 19 (2 cell lines at diagnosis and 15 cell lines at relapse), 26 (6 and 15), and 38 (11 and 15) cell lines, respectively. Of note, 14 BCP-ALL cell lines with the NR3C1 mutations were significantly more resistant to GC than those without mutations. In contrast, WHSC1 and CREBBP mutations were not associated with GC resistance. However, among the NR3C1 unmutated BCP-ALL cell lines, WHSC1 mutations tended to be associated with GC resistance and lower NR3C1 gene expression. Finally, we successfully established GC-resistant sublines of the GC-sensitive BCP-ALL cell line (697) by disrupting ligand binding and DNA binding domains of the NR3C1 gene using the CRISPR/Cas9 system. These observations demonstrated that somatic mutations of the NR3C1 gene, and possibly the WHSC1 gene, confer GC resistance in BCP-ALL.

A Novel RNA Synthesis Inhibitor, STK160830, Has Negligible DNA-Intercalating Activity for Triggering A p53 Response, and Can Inhibit p53-Dependent Apoptosis.

RNA synthesis inhibitors and protein synthesis inhibitors are useful for investigating whether biological events with unknown mechanisms require transcription or translation; however, the dependence of RNA synthesis has been difficult to verify because many RNA synthesis inhibitors cause adverse events that trigger a p53 response. In this study, we screened a library containing 9600 core compounds and obtained STK160830 that shows anti-apoptotic effects in irradiated wild-type-p53-bearing human T-cell leukemia MOLT-4 cells and murine thymocytes. In many of the p53-impaired cells and p53-knockdown cells tested, STK160830 did not show a remarkable anti-apoptotic effect, suggesting that the anti-apoptotic activity is p53-dependent. In the expression analysis of p53, p53-target gene products, and reference proteins by immunoblotting, STK160830 down-regulated the expression of many of the proteins examined, and the downregulation correlated strongly with its inhibitory effect on cell death. mRNA expression analyses by qPCR and nascent RNA capture kit revealed that STK160830 showed a decreased mRNA expression, which was similar to that induced by the RNA synthesis inhibitor actinomycin D but differed to some extent. Furthermore, unlike other RNA synthesis inhibitors such as actinomycin D, p53 accumulation by STK160830 alone was negligible, and a DNA melting-curve analysis showed very weak DNA-intercalating activity, indicating that STK160830 is a useful inhibitor for RNA synthesis without triggering p53-mediated damage responses.

HBO1-MLL interaction promotes AF4/ENL/P-TEFb-mediated leukemogenesis.

Leukemic oncoproteins cause uncontrolled self-renewal of hematopoietic progenitors by aberrant gene activation, eventually causing leukemia. However, the molecular mechanism underlying aberrant gene activation remains elusive. Here, we showed that leukemic MLL fusion proteins associate with the HBO1 histone acetyltransferase (HAT) complex through their trithorax homology domain 2 (THD2) in various human cell lines. MLL proteins associated with the HBO1 complex through multiple contacts mediated mainly by the ING4/5 and PHF16 subunits in a chromatin-bound context where histone H3 lysine 4 tri-methylation marks were present. Of the many MLL fusions, MLL-ELL particularly depended on the THD2-mediated association with the HBO1 complex for leukemic transformation. The C-terminal portion of ELL provided a binding platform for multiple factors including AF4, EAF1, and p53. MLL-ELL activated gene expression in murine hematopoietic progenitors by loading an AF4/ENL/P-TEFb (AEP) complex onto the target promoters wherein the HBO1 complex promoted the association with AEP complex over EAF1 and p53. Moreover, the NUP98-HBO1 fusion protein exerted its oncogenic properties via interaction with MLL but not its intrinsic HAT activity. Thus, the interaction between the HBO1 complex and MLL is an important nexus in leukemic transformation, which may serve as a therapeutic target for drug development.

HOXA9 promotes MYC-mediated leukemogenesis by maintaining gene expression for multiple anti-apoptotic pathways.

HOXA9 is often highly expressed in leukemias. However, its precise roles in leukemogenesis remain elusive. Here, we show that HOXA9 maintains gene expression for multiple anti-apoptotic pathways to promote leukemogenesis. In MLL fusion-mediated leukemia, MLL fusion directly activates the expression of MYC and HOXA9. Combined expression of MYC and HOXA9 induced leukemia, whereas single gene transduction of either did not, indicating a synergy between MYC and HOXA9. HOXA9 sustained expression of the genes implicated in the hematopoietic precursor identity when expressed in hematopoietic precursors, but did not reactivate it once silenced. Among the HOXA9 target genes, BCL2 and SOX4 synergistically induced leukemia with MYC. Not only BCL2, but also SOX4 suppressed apoptosis, indicating that multiple anti-apoptotic pathways underlie cooperative leukemogenesis by HOXA9 and MYC. These results demonstrate that HOXA9 is a crucial transcriptional maintenance factor that promotes MYC-mediated leukemogenesis, potentially explaining why HOXA9 is highly expressed in many leukemias.

Revertant somatic mosaicism as a cause of cancer.

Revertant (somatic) mosaicism is a spontaneous correction of a causative mutation in patients with congenital diseases. A relatively frequent event, revertant mosaicism may bring favorable outcomes that ameliorate disorders, and is therefore called "natural gene therapy." However, it has been revealed recently that "overcorrection" of inherited bone marrow failure in patients with sterile alpha motif domain containing 9 (SAMD9)/9L syndromes by revertant mosaicism induces myelodysplastic syndrome (MDS) with monosomy 7 that occasionally proceeds to acute myelogenous leukemia (AML). In this review, we interpret very complex mechanisms underlying MDS/AML in patients with SAMD9/9L syndromes. This includes multiple myeloid tumor suppressors on the long arm of chromosome 7, all of which act in a haploinsufficient fashion, and a difference in sensitivity to interferon between cells carrying a mutation and revertants. Overcorrection of mutants by somatic mosaicism is likely a novel mechanism in carcinogenesis.

Multiorgan failure with abnormal receptor metabolism in mice mimicking Samd9/9L syndromes.

Autosomal dominant sterile α motif domain containing 9 (Samd9) and Samd9L (Samd9/9L) syndromes are a large subgroup of currently established inherited bone marrow failure syndromes that includes myelodysplasia, infection, growth restriction, adrenal hypoplasia, genital phenotypes, and enteropathy (MIRAGE), ataxia pancytopenia, and familial monosomy 7 syndromes. Samd9/9L genes are located in tandem on chromosome 7 and have been known to be the genes responsible for myeloid malignancies associated with monosomy 7. Additionally, as IFN-inducible genes, Samd9/9L are crucial for protection against viruses. Samd9/9L syndromes are caused by gain-of-function mutations and develop into infantile myelodysplastic syndromes associated with monosomy 7 (MDS/-7) at extraordinarily high frequencies. We generated mice expressing Samd9LD764N, which mimic MIRAGE syndrome, presenting with growth retardation, a short life, bone marrow failure, and multiorgan degeneration. In hematopoietic cells, Samd9LD764N downregulates the endocytosis of transferrin and c-Kit, resulting in a rare cause of anemia and a low bone marrow reconstitutive potential that ultimately causes MDS/-7. In contrast, in nonhematopoietic cells we tested, Samd9LD764N upregulated the endocytosis of EGFR by Ship2 phosphatase translocation to the cytomembrane and activated lysosomes, resulting in the reduced expression of surface receptors and signaling. Thus, Samd9/9L is a downstream regulator of IFN that controls receptor metabolism, with constitutive activation leading to multiorgan dysfunction.

UTX maintains the functional integrity of the murine hematopoietic system by globally regulating aging-associated genes.

Epigenetic regulation is essential for the maintenance of the hematopoietic system, and its deregulation is implicated in hematopoietic disorders. In this study, UTX, a demethylase for lysine 27 on histone H3 (H3K27) and a component of COMPASS-like and SWI/SNF complexes, played an essential role in the hematopoietic system by globally regulating aging-associated genes. Utx-deficient (UtxΔ/Δ) mice exhibited myeloid skewing with dysplasia, extramedullary hematopoiesis, impaired hematopoietic reconstituting ability, and increased susceptibility to leukemia, which are the hallmarks of hematopoietic aging. RNA-sequencing (RNA-seq) analysis revealed that Utx deficiency converted the gene expression profiles of young hematopoietic stem-progenitor cells (HSPCs) to those of aged HSPCs. Utx expression in hematopoietic stem cells declined with age, and UtxΔ/Δ HSPCs exhibited increased expression of an aging-associated marker, accumulation of reactive oxygen species, and impaired repair of DNA double-strand breaks. Pathway and chromatin immunoprecipitation analyses coupled with RNA-seq data indicated that UTX contributed to hematopoietic homeostasis mainly by maintaining the expression of genes downregulated with aging via demethylase-dependent and -independent epigenetic programming. Of note, comparison of pathway changes in UtxΔ/Δ HSPCs, aged muscle stem cells, aged fibroblasts, and aged induced neurons showed substantial overlap, strongly suggesting common aging mechanisms among different tissue stem cells.

Activation of CpG-Rich Promoters Mediated by MLL Drives MOZ-Rearranged Leukemia.

Uncontrolled self-renewal of hematopoietic progenitors induces leukemia. To self-renew, leukemia cells must continuously activate genes that were previously active in their mother cells. Here, we describe the circuitry of a transactivation system responsible for oncogenic self-renewal. MLL recruits RNA polymerase II (RNAP2) to unmethylated CpG-rich promoters by its CXXC domain and activates transcription by transcriptional regulators, including the AF4 family/ENL family/P-TEFb complex, DOT1L, and p300/CBP histone acetyl transferases. MOZ also targets a broad range of CpG-rich promoters through association with RNAP2 and MLL. Leukemic fusion proteins such as MOZ-TIF2 and MLL-AFX constitutively activate CpG-rich promoters by aberrantly recruiting p300/CBP. Pharmacological inhibition of MLL or DOT1L induces differentiation of MOZ-TIF2-transformed cells. These results reveal that activation of unmethylated CpG-rich promoters mediated by MLL is the central mechanism of oncogenic self-renewal in MOZ-rearranged leukemia and indicate that the molecularly targeted therapies intended for MLL-rearranged leukemia can be applied for MOZ-rearranged leukemia.

Association between low doses of ionizing radiation, administered acutely or chronically, and time to onset of stroke in a rat model.

Exposure to high-doses of ionizing radiation has been reported to be associated with the risk of stroke. However, risks associated with lower dose exposures remain unclear, and there is little information available for the risk modification according to the dose-rate. There are few studies using animal models which might be able to provide complementary information on this association. In this study, the male stroke-prone spontaneously hypertensive rat (SHRSP) was used as a model animal. The rats were acutely irradiated with doses between 0 and 1.0 Gy or chronically irradiated with a cumulative dose of 0.5 or 1.0 Gy (at a dose rate of 0.05 or 0.1 Gy/day, respectively). The onset time of stroke related symptoms in SHRSP was used as an endpoint for evaluating the effects of low dose and the low dose-rate gamma-ray exposures. With respect to acute exposure, the time to the onset of stroke in the irradiated rats suggested the presence of a threshold around 0.1 Gy. For the low dose-rate chronically exposed, no significant increase in stroke symptom was observed. These findings are novel and demonstrate that the SHRSP system can be used to determine the association between the risk of stroke and radiation exposure with high sensitivity. Moreover, these studies provide important information regarding the association between the low dose and low dose-rate radiation exposure and circulatory diseases, especially stroke.