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B-cell acute lymphoblastic leukemia (B-ALL), a genetically heterogeneous disease, is classified into different molecular subtypes that are defined by recurrent gene rearrangements, gross chromosomal abnormalities, or specific gene mutations. Cells with these genetic alterations acquire a leukemia-initiating ability and show unique expression profiles. The distribution of B-ALL molecular subtypes is greatly dependent on age, which also affects treatment responsiveness and long-term survival, partly accounting for the inferior outcome in adolescents and young adults (AYA) and (older) adults with B-ALL. Recent advances in sequencing technology, especially RNA sequencing and the application of these technologies in large B-ALL cohorts have uncovered B-ALL molecular subtypes prevalent in AYA and adults. These new insights supply more precise estimations of prognoses and targeted therapies informed by sequencing results, as well as a deeper understanding of the genetic basis of AYA/adult B-ALL. This article provides an account of these technological advances and an overview of the recent major findings of B-ALL molecular subtypes in adults.

The cellular context that integrates gene expression, signaling, and metabolism dictates the oncogenic behavior and shapes the treatment responses in distinct cancer types. Although chimeric fusion proteins involving transcription factors (TF) are hallmarks of many types of acute lymphoblastic leukemia (ALL), therapeutically targeting the fusion proteins is a challenge. In this work, we characterize the core regulatory circuitry (CRC; interconnected autoregulatory loops of TFs) of B-ALL involving MEF2D-fusions and identify MEF2D-fusion and SREBF1 TFs as crucial CRC components. By gene silencing and pharmacologic perturbation, we reveal that the CRC integrates the pre-B-cell receptor (BCR) and lipid metabolism to maintain itself and govern malignant phenotypes. Small-molecule inhibitors of pre-BCR signaling and lipid biosynthesis disrupt the CRC and silence the MEF2D fusion in cell culture and show therapeutic efficacy in xenografted mice. Therefore, pharmacologic disruption of CRC presents a potential therapeutic strategy to target fusion protein–driven leukemia.Significance:Cancer type–specific gene expression is governed by transcription factors involved in a highly interconnected autoregulatory loop called CRC. Here, we characterized fusion protein–driven CRC and identified its pharmacologic vulnerabilities, opening therapeutic avenues to indirectly target fusion-driven leukemia by disrupting its CRC.See related commentary by Sadras and Müschen, p. 18.This article is highlighted in the In This Issue feature, p. 5

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Protection against subcutaneously inoculated OVA-expressing melanoma by OVA-peptide loaded iPS-pMC-DCs.

The use of dendritic cells (DC) to prime tumor-associated antigen-specific T-cell responses provides a promising approach to cancer immunotherapy. Embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) can differentiate into functional DCs, thus providing an unlimited source of DCs. However, the previously established methods of generating practical volumes of DCs from pluripotent stem cells (PSC) require a large number of PSCs at the start of the differentiation culture. In this study, we generated mouse proliferating myeloid cells (pMC) as a source of antigen-presenting cells (APC) using lentivirus-mediated transduction of the c-Myc gene into mouse PSC-derived myeloid cells. The pMCs could propagate almost indefinitely in a cytokine-dependent manner, while retaining their potential to differentiate into functional APCs. After treatment with IL4 plus GM-CSF, the pMCs showed impaired proliferation and differentiated into immature DC-like cells (pMC-DC) expressing low levels of major histocompatibility complex (MHC)-I, MHC-II, CD40, CD80, and CD86. In addition, exposure to maturation stimuli induced the production of TNFα and IL12p70, and enhanced the expression of MHC-II, CD40, and CD86, which is thus suggestive of typical DC maturation. Similar to bone marrow–derived DCs, they stimulated a primary mixed lymphocyte reaction. Furthermore, the in vivo transfer of pMC-DCs pulsed with H-2Kb-restricted OVA257-264 peptide primed OVA-specific cytotoxic T cells and elicited protection in mice against challenge with OVA-expressing melanoma. Overall, myeloid cells exhibiting cytokine-dependent proliferation and DC-like differentiation may be used to address issues associated with the preparation of DCs. Cancer Immunol Res; 3(6); 668–77. ©2015 AACR.

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Supplementary Materials and Methods

Supplementary Data. 1. Supplementary Methods 2. Supplementary Data Supplementary Table 1, Patient information at sampling. Supplementary Figure 1, A: Genomic alterations of both tumor and normal samples in Cases A-16, A-20, and A-35. B: Analysis of TCR-γ gene rearrangement in a case. Supplementary Figure 2, Re-expression of INK4a or ARF into ATL cell lines. Supplementary Table 2, Status of CD58 and B2M in ATL samples. Supplementary Table 3, Genetic alterations related to acute transformation of chronic type ATL. 3. References

Malignant mesothelioma (MM) is a rare but aggressive malignancy. Although the molecular genetics of MM are known, including BRCA1-associated protein-1 (BAP1) gene alterations, the prognosis of MM patients remains poor. Here we generated BAP1 knockout (BAP1-KO) human mesothelial cell clones to develop molecular-targeted therapeutics based on genetic alterations in MM. cDNA microarray and quantitative RT-PCR (qRT-PCR) analyses revealed high expression of a calcium/calmodulin-dependent protein kinase type II subunit delta (CAMK2D) gene in the BAP1-KO cells. CAMK2D was highly expressed in 70% of the human MM tissues (56/80) and correlated with the loss of BAP1 expression, making it a potential diagnostic and therapeutic target for BAP1-deficient MM. We screened an anticancer drugs library using BAP1-KO cells and successfully identified a CaMKII inhibitor, KN-93, which displayed a more potent and selective antiproliferative effect against BAP1-deficient cells than cisplatin or pemetrexed. KN-93 significantly suppressed the tumor growth in mice xenografted with BAP1-deficient MM cells. This study is the first to provide a potential molecular-targeted therapeutic approach for BAP1-deficient MM.

CEBPA-IGH, a fusion gene of the immunoglobulin heavy-chain locus (IGH) and the CCAAT enhancer-binding protein α (C/EBPα) gene, is recurrently found in B-ALL cases and causes aberrant expression of C/EBPα, a master regulator of granulocyte differentiation, in B cells. Forced expression of C/EBPα in B cells was reported to cause loss of B-cell identity due to the inhibition of Pax5, a master regulator of B-cell differentiation; however, it is not known whether the same mechanism is applicable for B-ALL development by CEBPA-IGH. It is known that a full-length isoform of C/EBPα, p42, promotes myeloid differentiation, whereas its N-terminal truncated isoform, p30, inhibits myeloid differentiation through the inhibition of p42; however, the differential role between p42 and p30 in ALL development has not been clarified. In the present study, we examined the effect of the expression of p42 and p30 in B cells by performing RNA-seq of mRNA from LCL stably transfected with p42 or p30. Unexpectedly, suppression of PAX5 target genes was barely observed. Instead, both isoforms suppressed the target genes of MEF2 family members (MEF2s), other regulators of B-cell differentiation. Similarly, MEF2s target genes rather than PAX5 target genes were suppressed in CEBP-IGH-positive ALL (n = 8) compared with other B-ALL (n = 315). Furthermore, binding of both isoforms to MEF2s target genes and the reduction of surrounding histone acetylation were observed in ChIP-qPCR. Our data suggest that the inhibition of MEF2s by C/EBPα plays a role in the development of CEBPA-IGH-positive ALL and that both isoforms work co-operatively to achieve it.

There has been a great deal of research on cell division and its mechanisms; however, its processes have not yet been entirely elucidated. To find novel proteins that regulate cell division, we performed the screening using siRNAs and/or the expression plasmid of the target genes and identified leucine zipper protein 1 (LUZP1). Recent studies have shown that LUZP1 interacts with various proteins and stabilizes the actin cytoskeleton; however, the function of LUZP1 in mitosis is not known. In this study, we found that LUZP1 colocalized with the chromosomal passenger complex (CPC) at the centromere in metaphase and at the central spindle in anaphase and that these LUZP1 localizations were regulated by CPC activity and kinesin family member 20A (KIF20A). Mass spectrometry analysis identified that LUZP1 interacted with death-associated protein kinase 3 (DAPK3), one regulator of the cleavage furrow ingression in cytokinesis. In addition, we found that LUZP1 also interacted with myosin light chain 9 (MYL9), a substrate of DAPK3, and comprehensively inhibited MYL9 phosphorylation by DAPK3. In line with a known role for MYL9 in the actin-myosin contraction, LUZP1 suppression accelerated the constriction velocity at the division plane in our timelapse analysis. Our study indicates that LUZP1 is a novel regulator for cytokinesis that regulates the constriction velocity of the contractile ring.

Internal tandem duplication (ITD) of FMS-like tyrosine kinase 3 (FLT3) confers poor prognosis and is found in approximately 25% of cases of acute myeloid leukemia (AML). Although FLT3 inhibitors have shown clinical benefit in patients with AML harboring FLT3-ITD, the therapeutic effect is limited. Here, to explore alternative therapeutics, we established a cellular model of monoallelic FLT3ITD/WT cells using the CRISPR-Cas9 system in a human myeloid leukemia cell line, K562. cDNA microarray analysis revealed elevated CD52 expression in K562–FLT3ITD/WT cells compared to K562–FLT3WT/WT cells, an observation that was further confirmed by quantitative real-time-PCR and flow cytometric analyses. The elevated expression of CD52 in K562–FLT3ITD/WT cells was decreased in wild-type FLT3 (FLT3-WT) knock-in K562–FLT3ITD/WT cells. In K562–FLT3ITD/WT cells, a STAT5 inhibitor, pimozide, downregulated CD52 protein expression while an AKT inhibitor, afuresertib, did not affect CD52 expression. Notably, an anti-CD52 antibody, alemtuzumab, induced significant antibody-dependent cell-mediated cytotoxicity (ADCC) in K562-FLT3ITD/WT cells compared to K562–FLT3WT/WT cells. Furthermore, alemtuzumab significantly suppressed the xenograft tumor growth of K562–FLT3ITD/WT cells in severe combined immunodeficiency (SCID) mice. Taken together, our data suggested that genetically modified FLT3-ITD knock-in human myeloid leukemia K562 cells upregulated CD52 expression via activation of STAT5, and alemtuzumab showed an antitumor effect via induction of ADCC in K562–FLT3ITD/WT cells. Our findings may allow establishment of a new therapeutic option, alemtuzumab, to treat leukemia with the FLT3-ITD mutation.

Malignant pleural mesothelioma (MPM) is an aggressive malignancy of the pleura that is currently incurable due to the lack of an effective early diagnostic method and specific medication. The CDKN2A (p16) and NF2 genes are both frequently mutated in MPM. To understand how these mutations contribute to MPM tumor growth, we generated NF2/p16 double-knockout (DKO) cell clones using human MeT-5A and HOMC-B1 mesothelial cell lines. Cell growth and migration activities were significantly increased in DKO compared with parental cells. cDNA microarray analysis revealed differences in global gene expression profiles between DKO and parental cells. Quantitative PCR and western blot analyses showed upregulation of CD24 concomitant with increased phosphorylation of AKT, p70S6K, and c-Jun in DKO clones. This upregulation was abrogated by exogenous expression of NF2 and p16. CD24 knockdown in DKO cells significantly decreased TGF-β1 expression and increased expression of E-cadherin, an epithelial–mesenchymal transition marker. CD24 was highly expressed in human mesothelioma tissues (28/45 cases, 62%) and associated with the loss of NF2 and p16. Public data analysis revealed a significantly shorter survival time in MPM patients with high CD24 gene expression levels. These results strongly indicate the potential use of CD24 as a prognostic marker as well as a novel diagnostic and therapeutic target for MPM.

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is intractable and mostly harbors genetic alterations that activate JAK or ABL signaling. The commonest subtype of Ph-like ALL exhibits the CRLF2 gene arrangement that brings about JAK1/2-STAT5 pathway activation. However, JAK1/2 inhibition alone is insufficient for treatment, necessitating combinatorial therapies targeting multiple signals. To better understand the mechanisms underlying the insufficient efficacy of JAK inhibition, this study explored gene expression changes upon treatment with a JAK1/2 inhibitor (ruxolitinib) and revealed BCL6 elevation as one such mechanism. Upregulated BCL6 suppressed TP53 expression along with its downstream cell cycle inhibitor p21 (CDKN2A) and proapoptotic molecules, such as FAS, TNFRSF10B, BID, BAX, BAK, PUMA, and NOXA, conferring cells in part with therapy resistance. The BCL6 inhibition (FX1) alone was able to upregulate TP53 and restore TP53 expression that ruxolitinib had diminished. In addition, ruxolitinib and FX1 concertedly downregulated MYC. As a result, FX1 treatment alone had growth-inhibitory and apoptosis-sensitizing effects, but the combination of ruxolitinib and FX1 more potently inhibited leukemia cell growth, enhanced apoptosis sensitivity, and prolonged xenografted mice survival. These findings provide one mechanism for the insufficiency of JAK inhibition for CRLF2-rearranged ALL treatment and BCL6 inhibition as a potentially helpful adjunctive therapy combined with JAK inhibition.

Ameloblastoma is a rare odontogenic benign tumor accounting for less than 1% of head and neck tumors. Advanced next generation sequencing (NGS) analyses identified high frequency of BRAF V600E and SMO L412F mutations in ameloblastoma. Despite the existence of whole genomic sequence information from patients with ameloblastoma, entire molecular signature of and the characteristics of ameloblastoma cells are still obscure. In this study, we sought to uncover the molecular basis of ameloblastoma and to determine the cellular phenotype of ameloblastoma cells with BRAF mutations. Our comparative cDNA microarray analysis and gene set enrichment analysis (GSEA) showed that ameloblastoma exhibited a distinct gene expression pattern from the normal tissues: KRAS‐responsive gene set is significantly activated in ameloblastoma. Importantly, insulin like growth factor 2 (IGF2), a member of KRAS‐responsive genes, enhances the proliferation of an ameloblastoma cell line AMU‐AM1 with BRAF mutation. In addition, Toll‐like receptor 2 (TLR2) knockdown readily inactivated KRAS‐responsive gene sets as well as increases caspase activities, suggesting that TLR2 signaling may mediate cell survival signaling in ameloblastoma cells. Collectively, the findings may help to further clarify the pathophysiology of ameloblastoma and lead to the development of precision medicine for patients with ameloblastoma., The present study is the first to demonstrate novel molecular characteristics in ameloblastoma, where oncogenic gene signatures including KRAS‐responsive genes and EGFR‐related genes were significantly activated. Our findings may help to further clarify the pathophysiology of ameloblastoma and lead to the development of precision medicine for patients with ameloblastoma.

Loss of heterozygosity or mutation of the family with sequence similarity 46, member C (FAM46C) gene on chromosome band 1p12 is associated with shorter overall survival of patients with multiple myeloma (MM). In this study, using human MM cell lines (KMS‐11, OCI‐My5, and ANBL‐6), we generated FAM46C−/− cell clones and examined the effect of disruption of FAM46C on cell survival and cellular signaling. Cell proliferation assays showed increased clonogenicity of FAM46C−/− KMS‐11 cells compared to WT cells. Xenograft experiments showed significantly shorter overall survival of mice harboring the FAM46C−/− cell‐derived tumors than mice with the FAM46CWT cell‐derived tumors. Notably, levels of phosphorylated Akt and its substrates increased both in vitro and in vivo in the FAM46C−/− cells compared to WT cells. In addition, caspase activities decreased in the FAM46C−/− cells. Results of gene set enrichment analysis showed that loss of FAM46C significantly activated serum‐responsive genes while inactivating phosphatase and tensin homolog (PTEN)‐related genes. Mechanistically, loss of FAM46C decreased the PTEN activity, number of apoptotic cells, and caspase activities. PF‐04691502, a selective PI3K inhibitor, suppressed the augmented phosphorylation of Akt and its substrate FoxO3a. Treatment with afuresertib (a specific Akt inhibitor) in combination with bortezomib additively decreased FAM46C−/− MM cell survival. Collectively, this study is the first to report that loss of FAM46C triggers the concomitant activation of the PI3K‐Akt signaling pathway, which might be a therapeutic target for MM with abnormalities in the FAM46C gene., This study shows, for the first time, that FAM46C loss promotes the PI3K‐Akt signaling pathway in multiple myeloma cells. Notably, loss of FAM46C sensitized cells to specific inhibitor of PI3K‐Akt, afuresertib.

Targeted knock-in supported by the CRISPR/Cas systems enables the insertion, deletion, and substitution of genome sequences exactly as designed. Although this technology is considered to have wide range of applications in life sciences, one of its prerequisites for practical use is to improve the efficiency, precision, and specificity achieved. To improve the efficiency of targeted knock-in, there first needs to be a reporter system that permits simple and accurate monitoring of targeted knock-in events. In the present study, we created such a system using the PIGP gene, an autosomal gene essential for GPI-anchor biosynthesis, as a reporter gene. We first deleted a PIGP allele using Cas9 nucleases and then incorporated a truncating mutation into the other PIGP allele in two near-diploid human cell lines. The resulting cell clones were used to monitor the correction of the PIGP mutations by detecting GPI anchors distributed over the cell membrane via flow cytometry. We confirmed the utility of these reporter clones by performing targeted knock-in in these clones via a Cas9 nickase-based strategy known as tandem paired nicking, as well as a common process using Cas9 nucleases, and evaluating the efficiencies of the achieved targeted knock-in. We also leveraged these reporter clones to test a modified procedure for tandem paired nicking and demonstrated a slight increase in the efficiency of targeted knock-in by the new procedure. These data provide evidence for the utility of our PIGP-based assay system to quantify the efficiency of targeted knock-in and thereby help improve the technology of targeted knock-in.

Targeted knock-in assisted by the CRISPR/Cas9 system is an advanced technology with promising applications in various research fields including medical and agricultural sciences. However, improvements in the efficiency, precision, and specificity of targeted knock-in are prerequisites to facilitate the practical application of this technology. To improve the efficiency of targeted knock-in, it is necessary to have a molecular system that allows sensitive monitoring of targeted knock-in events with simple procedures.We developed an assay, named CD55 correction assay, with which to monitor CD55 gene correction accomplished by targeted knock-in. To create the reporter clones used in this assay, we initially introduced a 7.7-kb heterozygous deletion covering CD55 exons 2-5, and then incorporated a truncating mutation within exon 4 of the remaining CD55 allele in human cell lines. The resultant reporter clones that lost the CD55 protein on the cell membrane were next transfected with Cas9 constructs along with a donor plasmid carrying wild-type CD55 exon 4. The cells were subsequently stained with fluorescence-labeled CD55 antibody and analyzed by flow cytometry to detect CD55-positive cells. These procedures allow high-throughput, quantitative detection of targeted gene correction events occurring in an endogenous human gene.The current study demonstrated the utility of the CD55 correction assay to sensitively quantify the efficiency of targeted knock-in. When used with the PIGA correction assay, the CD55 correction assay will help accurately determine the efficiency of targeted knock-in, precluding possible experimental biases caused by cell line-specific and locus-specific factors.

Tandem paired nicking (TPN) is a method of genome editing that enables precise and relatively efficient targeted knock-in without appreciable restraint by p53-mediated DNA damage response. TPN is initiated by introducing two site-specific nicks on the same DNA strand using Cas9 nickases in such a way that the nicks encompass the knock-in site and are located within a homologous region between a donor DNA and the genome. This nicking design results in the creation of two nicks on the donor DNA and two in the genome, leading to relatively efficient homology-directed recombination between these DNA fragments. In this study, we sought to identify the optimal design of TPN experiments that would improve the efficiency of targeted knock-in, using multiple reporter systems based on exogenous and endogenous genes. We found that efficient targeted knock-in via TPN is supported by the use of 1700–2000-bp donor DNAs, exactly 20-nt-long spacers predicted to be efficient in on-target cleavage, and tandem-paired Cas9 nickases nicking at positions close to each other. These findings will help establish a methodology for efficient and precise targeted knock-in based on TPN, which could broaden the applicability of targeted knock-in to various fields of life science.

The genetic basis of leukemogenesis in adults with B-cell acute lymphoblastic leukemia (B-ALL) is largely unclear, and its clinical outcome remains unsatisfactory. This study aimed to advance the understanding of biological characteristics, improve disease stratification, and identify molecular targets of adult B-ALL. Adolescents and young adults (AYA) (15 to 39 years old, n = 193) and adults (40 to 64 years old, n = 161) with Philadelphia chromosome-negative (Ph−) B-ALL were included in this study. Integrated transcriptomic and genetic analyses were used to classify the cohort into defined subtypes. Of the 323 cases included in the RNA sequencing analysis, 278 (86.1%) were classified into 18 subtypes. The ZNF384 subtype (22.6%) was the most prevalent, with 2 novel subtypes (CDX2-high and IDH1/2-mut) identified among cases not assigned to the established subtypes. The CDX2-high subtype (3.4%) was characterized by high expression of CDX2 and recurrent gain of chromosome 1q. The IDH1/2-mut subtype (1.9%) was defined by IDH1 R132C or IDH2 R140Q mutations with specific transcriptional and high-methylation profiles. Both subtypes showed poor prognosis and were considered inferior prognostic factors independent of clinical parameters. Comparison with a previously reported pediatric B-ALL cohort (n = 1003) showed that the frequencies of these subtypes were significantly higher in AYA/adults than in children. We delineated the genetic and transcriptomic landscape of adult B-ALL and identified 2 novel subtypes that predict poor disease outcomes. Our findings highlight the age-dependent distribution of subtypes, which partially accounts for the prognostic differences between adult and pediatric B-ALL.

Zinc-finger protein 384 (ZNF384) fusion (Z-fusion) genes have recently been identified as recurrent fusion genes in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) and have been detected in 7-17% of Philadelphia chromosome-negative BCP-ALL cases. We selected SALL4 and ID2 as potential Z-fusion-specific transcriptional targets that might lead to the differentiation disorder of Z-fusion-positive ALL. The introduction of EP300-ZNF384 and SYNRG-ZNF384 induced the expression of these genes. Z-fusion proteins exhibited stronger transcriptional activities on the promoter or enhancer region of these genes than Wild-Z. Furthermore, GST pull-down assay revealed that Z-fusion proteins associated more strongly with EP300 than Wild-Z. Coexpression of EP300 specifically enhanced the transcriptional activities of Z-fusion proteins. We propose the increased EP300 binding of Z-fusion proteins as a mechanism for their increased transcriptional activities.

Malignant pleural mesothelioma (MPM), a highly refractory tumor, is currently incurable due to the lack of an early diagnosis method and medication, both of which are urgently needed to improve the survival and/or quality of life of patients. NF2 is a tumor suppressor gene and is frequently mutated in MPM. Using a CRISPR/Cas9 system, we generated an NF2-knockout human mesothelial cell line, MeT-5A (NF2-KO). In NF2-KO cell clones, cell growth, clonogenic activity, migration activity, and invasion activity significantly increased compared with those in NF2-WT cell clones. Complementary DNA microarray analysis clearly revealed the differences in global gene expression profile between NF2-WT and NF2-KO cell clones. Quantitative PCR analysis and western blot analysis showed that the upregulation of fibroblast growth factor receptor 2 (FGFR2) was concomitant with the increases in phosphorylation levels of JNK, c-Jun, and retinoblastoma (Rb) in NF2-KO cell clones. These increases were all abrogated by the exogenous expression of NF2 in the NF2-KO clone. In addition, the disruption of FGFR2 in the NF2-KO cell clone suppressed cell proliferation as well as the phosphorylation levels of JNK, c-Jun, and Rb. Notably, FGFR2 was found to be highly expressed in NF2-negative human mesothelioma tissues (11/12 cases, 91.7%) but less expressed in NF2-positive tissues. Collectively, these findings suggest that NF2 deficiency might play a role in the tumorigenesis of human mesothelium through mediating FGFR2 expression; FGFR2 would be a candidate molecule to develop therapeutic and diagnostic strategies for targeting MPM with NF2 loss.

MEF2D-fusion (M-fusion) genes are newly discovered recurrent gene abnormalities that are detected in approximately 5 % of acute lymphoblastic leukemia (ALL) cases. Their introduction to cells has been reported to transform cell lines or increase the colony formation of bone marrow cells, suggesting their survival-supporting ability, which prompted us to examine M-fusion-targeting drugs. To identify compounds that reduce the protein expression level of MEF2D, we developed a high-throughput screening system using 293T cells stably expressing a fusion protein of MEF2D and luciferase, in which the protein expression level of MEF2D was easily measured by a luciferase assay. We screened 3766 compounds with known pharmaceutical activities using this system and selected staurosporine as a potential inducer of the proteolysis of MEF2D. Staurosporine induced the proteolysis of M-fusion proteins in M-fusion (+) ALL cell lines. Proteolysis was inhibited by caspase inhibitors, not proteasome inhibitors, suggesting caspase dependency. Consistent with this result, the growth inhibitory effects of staurosporine were stronger in M-fusion (+) ALL cell lines than in negative cell lines, and caspase inhibitors blocked apoptosis induced by staurosporine. We identified the cleavage site of MEF2D-HNRNPUL1 by caspases and confirmed that its caspase cleavage-resistant mutant was resistant to staurosporine-induced proteolysis. Based on these results, we investigated another Food and Drug Administration-approved caspase activator, venetoclax, and found that it exerted similar effects to staurosporine, namely, the proteolysis of M-fusion proteins and strong growth inhibitory effects in M-fusion (+) ALL cell lines. The present study provides novel insights into drug screening strategies and the clinical indications of venetoclax.

[Figure: see text] Multiple myeloma (MM) remains an intractable hematological malignancy, despite recent advances in anti-MM drugs. Here, we show that role of PDZ binding kinase (PBK) in MM tumor growth. We identified that interleukin-6 (IL-6) readily increases PBK expression. Kaplan-Meier analysis showed that the MM patients with higher expression of PBK have a significant shorter survival time compared with those with moderate/lower expression of PBK. Knockout of PBK dramatically suppressed in vivo tumor growth in MM cells, while genome editing of PBK changing from asparagine to serine substitution (rs3779620) slightly suppresses the tumor formation. Mechanistically, loss of PBK increased the number of apoptotic cells with concomitant decrease in the phosphorylation level of Stat3 as well as caspase activities. A novel PBK inhibitor OTS514 significantly decreased KMS-11-derived tumor growth. These findings highlight the novel oncogenic role of PBK in tumor growth of myeloma, and it might be a novel therapeutic target for the treatment of patients with MM.

Summary: Targeted knockin mediated by double-stranded DNA cleavage is accompanied by unwanted insertions and deletions (indels) at on-target and off-target sites. A nick-mediated approach scarcely generates indels but exhibits reduced efficiency of targeted knockin. Here, we demonstrate that tandem paired nicking, a method for targeted knockin involving two Cas9 nickases that create nicks at the homologous regions of the donor DNA and the genome in the same strand, scarcely creates indels at the edited genomic loci, while permitting the efficiency of targeted knockin largely equivalent to that of the Cas9-nuclease-based approach. Tandem paired nicking seems to accomplish targeted knockin by DNA recombination analogous to Holliday’s model and creates intended genomic changes without introducing additional nucleotide changes, such as silent mutations. Targeted knockin through tandem paired nicking neither triggers significant p53 activation nor occurs preferentially in p53-suppressed cells. These properties of tandem paired nicking demonstrate its utility in precision genome engineering. : Hyodo et al. describe a method of precise genome editing using Cas9 nickases, which barely introduces unwanted insertions and deletions at the edited genomic loci. This method neither activates nor is inhibited by p53 signalling, allowing safe genome editing and suggesting promise for this method in clinical genome engineering. Keywords: CRISPR/Cas9, nickase, tandem paired nicking, knockin, Holliday’s model, crossover, non-crossover, p53, p21, tandem nicking

Malignant pleural mesothelioma (MPM), an asbestos‐related occupational disease, is an aggressive and incurable tumor of the thoracic cavity. Despite recent advances in MPM treatment, overall survival of patients with MPM is very low. Recent studies have implicated that PI3K/Akt signaling is involved in MPM cell survival and development. To investigate the effects of Akt inhibitors on MPM cell survival, we examined the effects of nine selective Akt inhibitors, namely, afuresertib, Akti‐1/2, AZD5363, GSK690693, ipatasertib, MK‐2206, perifosine, PHT‐427, and TIC10, on six MPM cell lines, namely, ACC‐MESO‐4, Y‐MESO‐8A, MSTO‐211H, NCI‐H28, NCI‐H290, and NCI‐H2052, and a normal mesothelial cell line MeT‐5A. Comparison of IC 50 values of the Akt inhibitors showed that afuresertib, an ATP‐competitive specific Akt inhibitor, exerted tumor‐specific effects on MPM cells. Afuresertib significantly increased caspase‐3 and caspase‐7 activities and apoptotic cell number among ACC‐MESO‐4 and MSTO‐211H cells. Moreover, afuresertib strongly arrested the cell cycle in the G1 phase. Western blotting analysis showed that afuresertib increased the expression of p21WAF 1/ CIP 1 and decreased the phosphorylation of Akt substrates, including GSK‐3β and FOXO family proteins. These results suggest that afuresertib‐induced p21 expression promotes G1 phase arrest by inducing FOXO activity. Furthermore, afuresertib significantly enhanced cisplatin‐induced cytotoxicity. Interestingly, results of gene set enrichment analysis showed that afuresertib modulated the expression E2F1 and MYC, which are associated with fibroblast core serum response. Together, these results suggest that afuresertib is a useful anticancer drug for treating patients with MPM.

MEF2D fusion (M-fusion) genes are newly discovered recurrent gene abnormalities that are detected in approximately 5% of acute lymphoblastic leukemia (ALL) cases. We previously found that the loss of the micro RNA target site in wild-type MEF2D gene by translocation led to strong expression of M-fusion protein in ALL cells by evasion from micro RNA and that M-fusion protein inhibited the transcriptional activity of PAX5, a B-cell differentiation regulator, in a dose-dependent manner. These findings prompted us to explore drugs that induced proteolysis of M-fusion protein as possible therapeutic agents for M-fusion-positive ALL. We developed a high-throughput screening system to find compounds that reduced protein expression level of MEF2D. The expression vector of the fusion protein of N-terminal half of MEF2D (MEF2D N) and luciferase (MEF2D N-Luc) was stably transfected to 293T cells (MEF2D N-Luc/293T). Stable transfectant of the expression vector of luciferase was also established (Luc/293T). We could easily measure protein expression level in these cells by luciferase assay. We screened 3766 compounds with known pharmaceutical activities with this system and selected staurosporine, a multi-kinase inhibitor, as a possible proteolysis-inducer of MEF2D. Staurosporine strongly reduced the luciferase value in MEF2D N-Luc/293T but not in Luc/293T (Figure 1A). Staurosporine induced proteolysis of MEF2D-HNRNPUL1 (M-H) and MEF2D-DAZAP1 (M-D) in M-fusion-positive ALL cell lines within 6 h. Proteolysis of M-fusion proteins were inhibited not by MG-132, a proteasome inhibitor, but by Z-VAD FMK, a caspase inhibitor, indicating that these proteolyses were caspase-dependent (Figure 1B). Consistent with this, Z-VAD-FMK blocked apoptosis by staurosporine in M-H positive ALL cell lines . We confirmed the cleavage of M-H by caspase 3 and caspase 7 in vitro and identified the cleavage site (Figure 1C). Furthermore, staurosporine demonstrated stronger cytotoxic effect on M-fusion-positive ALL cell lines than M-fusion-negative ones (Figure 1D). These results indicated that staurosporine induced apoptosis of M-fusion-positive ALL cells through caspase-dependent proteolysis of M-fusion protein at least in part. Luciferase-based proteolysis screening provided a novel strategy for the development of anti-cancer drugs. Figure legends Figure 1. A. Staurosporine strongly reduced the luciferase value in MEF2D N-Luc/293T but not in Luc/293T. MEF2D N-Luc/293T were treated with 3766 compounds (2uM each) for 24 h. Then luciferase assays were performed to estimate the amount of MEF2D N-Luc protein. Top 15 compounds which reduced the relative luciferase value were selected for the second screening where compounds were added to MEF2D N-Luc/293T and Luc/293T, then we estimated their effect on the expression of MEF2D N-Luc and luciferase. Results of the second screening were plotted on a scattergram, on which the relative luciferase value in Luc/293T and MEF2D N-Luc/293T were set on the Y-axis and X-axis, respectively. Relative luciferase values are relative values to those of control cells treated with vehicle (DMSO). Staurosporine and K252a, an analog of staurosporine, were selected as hit compounds which reduced MEF2D N-Luc but not luciferase. B. Staurosporine reduced the expression of M-H and M-D, which inhibited by Z-VAD FMK. M-H-positive ALL cell lines, Kasumi-7 and Kasumi-9 and a M-D-positive ALL cell line, TS-2 were treated with 1uM staurosporine, 20uM Z-VAD-FMK, or both for 6 h. Cells were lysed and subjected to immunoblotting with indicated antibodies. C. Caspase-3 and caspase-7 cleaved M-H in vitro. M-H and its mutants with aspartate substitutions at possible caspase cleavage sites were synthesized with [35S]-methionine-labeled in vitro translation and were incubated with purified caspase-3 or -7. Cleaved fragments were resolved on SDS-PAGE and visualized by autoradiography. Mutant 4 was resistant to the cleavage by both caspases. D. Staurosporine demonstrated stronger cytotoxic effect on M-fusion-positive ALL cell lines than M-fusion-negative ones. ALL cell lines were treated with staurosporine at the indicated doses for 24 h. Cell viabilities were measured using MTT assay. Figure 1 Disclosures Kiyoi: Takeda Pharmaceutical Co., Ltd.: Research Funding; Pfizer Japan Inc.: Honoraria; Astellas Pharma Inc.: Honoraria, Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; FUJIFILM Corporation: Research Funding; Eisai Co., Ltd.: Research Funding; Bristol-Myers Squibb: Research Funding; Daiichi Sankyo Co., Ltd: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Research Funding; Perseus Proteomics Inc.: Research Funding.