ALBERT

Description: Additional sex combs-like 1 (ASXL1), an epigenetic modulator, is frequently mutated in myeloid neoplasms. Recent analyses of mutant ASXL1 conditional knock-in (ASXL1-MT-KI) mice suggested that ASXL1-MT alone is insufficient for myeloid transformation. In our previous study, we utilized retrovirus-mediated insertional mutagenesis, which exhibited susceptibility of ASXL1-MT-KI hematopoietic cells to transform into myeloid leukemia cells. In this screening, we identified Hematopoietically expressed homeobox (HHEX) gene as one of the common retrovirus integration sites. In this study, we investigated the potential cooperation between ASXL1-MT and HHEX in myeloid leukemogenesis. Expression of HHEX enhanced proliferation of ASXL1-MT expressing HSPCs by inhibiting apoptosis and blocking differentiation, whereas it showed only modest effect in normal HSPCs. Moreover, ASXL1-MT and HHEX accelerated the development of RUNX1-ETO9a and FLT3-ITD leukemia. Conversely, HHEX depletion profoundly attenuated the colony-forming activity and leukemogenicity of ASXL1-MT-expressing leukemia cells. Mechanistically, we identified MYB and ETV5 as downstream targets for ASXL1-MT and HHEX by using transcriptome and ChIP-seq analyses. Moreover, we found that expression of ASXL1-MT enhanced the binding of HHEX to the promoter loci of MYB or ETV5 via reducing H2AK119ub. Depletion of MYB or ETV5 induced apoptosis or differentiation in ASXL1-MT-expressing leukemia cells, respectively. In addition, ectopic expression of MYB or ETV5 reversed the reduced colony-forming activity of HHEX-depleted ASXL1-MT-expressing leukemia cells. These findings indicated that the HHEX-MYB/ETV5 axis promotes myeloid transformation in ASXL1-mutated preleukemia cells.

Description: Defining the epigenetic mechanisms (e.g. chromatin modifications) that underlie T cell fate decisions is a major challenge. The transcriptional coactivators CREB binding protein (CBP) and the closely related p300 comprise a two-member family of histone/protein acetyltransferases that interact with over 50 T lymphocyte-essential transcriptional regulators. Rather than having distinct regulatory roles, CBP and p300 are often thought to confer utilitarian transactivation and histone modifying functions to transcription factors that mediate T cell fate. In contrast to this view, we show here that CBP acts uniquely in conventional T cell development. Inactivation of CBP, but not p300, starting at the double negative stage of T cell development yielded thymocytes with partial activation of an effector/memory- or innate-T cell program. CD8SP thymocytes from CBP mutant mice expressed genes that define professional CD8 cells such as Il-2/Il-15 receptor β chain, granzyme A, interferon γ (Ifnγ), Fas ligand, perforin, and the chemokine receptors Ccr5, and Cxcr3. CD4SP thymocytes from CBP mutant mice also expressed effector genes such as Ifnγ, Il-4, and Ccr5. In addition, CD8SP and CD4SP thymocytes from CBP mutant mice produced Ifnγ protein when the cells were stimulated with phorbol ester and ionomycin. Mechanistically, loss of CBP acted cell non-autonomously to induce the expression of the CD8 T cell master regulatory transcription factor eomesodermin (Eomes). This suggests that CBP in thymocytes or T cells controls an extracellular factor that helps demarcate conventional naïve T cell development in the thymus from effector/memory T cell differentiation in the periphery.

Description: Paraspeckles are sub-nuclear structures found in the interchromatin space of mammalian cells. The core paraspeckle components include a lncRNA NEAT1 and members of the DBHS family of proteins: NONO, SFPQ, and PSPC1. Paraspeckles and their components play diverse roles in gene regulatory networks, including transcription, alternative RNA splicing, nuclear retention of RNA, and DNA repair. Although a previous study showed the presence of paraspeckles in hematopoietic stem and progenitor cells (HSPCs), their roles in normal and malignant hematopoiesis remain largely unknown. ASXL1 regulates gene expression through interactions with multiple epigenetic regulators. Somatic mutations in ASXL1 gene occur frequently in myeloid neoplasms. We previously generated a hematopoietic lineage-specific conditional knockin (KI) mouse of a C-terminally truncated form of ASXL1-mutant (ASXL1-MT), and showed that ASXL1-MT inhibited repopulating capability of HSPCs. We performed deep RNA sequencing using HSPCs from ASXL1-MT-KI mice, and found aberrant alternative splicing in multiple genes involved in hematopoiesis. The altered splicing in ASXL1-MT-KI HSPCs included abnormal exon skipping or retention in Runx1, Traf6, Atm, and Dnmt3b. These findings, together with a previous report showing that ASXL1 mutations affect alternative splicing in U937 cells, strongly indicate the involvement of ASXL1 in RNA splicing machinery. Because a previous interactome analysis suggested the association between NONO and ASXL1, we hypothesized that ASXL1 may play a role in RNA maturation processes through interactions with paraspeckle proteins. To test this hypothesis, we examined physical and functional interactions between paraspeckle components and ASXL1. We found that both wild-type and mutant ASXL1 interact with NONO and SFPQ in 293T cells. Interestingly, protein and RNA immunoprecipitation (RIP) analyses revealed that coexpression of wild-type ASXL1, but not mutant ASXL1, enhanced interactions between NONO and histone H3 as well as NONO and NEAT1. These results suggest that ASXL1 acts as a scaffolding protein that assembles paraspeckle proteins and histones to promote transcription and RNA processing. Importantly, mutant ASXL1 loses this function. Next, we assessed subcellular localization of Nono in HSPCs from control and ASXL1-MT-KI mice. We observed predominant cytoplasmic expression of Nono in ASXL1-MT KI HSPCs, while Nono mainly localized in the nucleus in control cells (Figure 1). In addition, expression of NEAT1_2 isoform, which is essential for paraspeckle formation and maintenance, was substantially downregulated in ASXL1-MT-KI HSPCs. Consistent with these observations, RNA FISH against NEAT1 and immunofluorescence against NONO revealed disrupted paraspeckle formation in ASXL1-MT-KI HSPCs. These data suggest that ASXL1-MT promotes nuclear export of Nono, which results in disruption of paraspeckles in HSPCs. NONO has nuclear localization signal (NLS) at its C-terminus, and it was previously shown that a cytoplasmic C-truncated form of NONO induced senescence in fibroblasts. To assess the effect of forced expression of the cytoplasmic NONO in hematopoietic cells, we transduced vector or a NONO mutant lacking the NLS domain (NONO-ΔNLS) into c-Kit+ bone marrow cells, and transplanted these cells into recipient mice. NONO-ΔNLS induced overproduction of reactive oxygen species (ROS) and reduced engraftment of bone marrow progenitors as ASXL1-MT did. We then assessed the effect of Nono depletion in ASXL1-MT-KI HSPCs using CRISPR/Cas9 system. We crossed ASXL1-MT-KI mice with Rosa26-LSL-Cas9-KI mice, and c-Kit+ bone marrow cells derived from these mice were transduced with a non-targeting or Nono-targeting sgRNAs. This experiment revealed that Nono depletion reverted the impaired repopulation of ASXL1-MT-KI HSPCs after transplantation. Taken together, these data indicate that the cytoplasmic localization of Nono induced by ASXL1-MT has the negative impact on HSPC function. In summary, this study reveals a novel link between an epigenetic regulator ASXL1 and paraspeckle formation. The aberrant interaction between mutant ASXL1 and NONO results in NONO mislocalization, paraspeckle disruption and HSPC dysfunction. Our findings also suggest potentially important roles for paraspeckles to maintain normal hematopoiesis. Disclosures Ogawa: Qiagen Corporation: Patents & Royalties; RegCell Corporation: Equity Ownership; Asahi Genomics: Equity Ownership; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Kan Research Laboratory, Inc.: Consultancy.

Description: CREB-binding protein (CBP) and its para-log p300 are transcriptional coactivators that physically or functionally interact with over 320 mammalian and viral proteins, including 36 that are essential for B cells in mice. CBP and p300 are generally considered limiting for transcription, yet their roles in adult cell lineages are largely unknown since homozygous null mutations in either gene or compound heterozygosity cause early embryonic lethality in mice. We tested the hypotheses that CBP and p300 are limiting and that each has unique properties in B cells, by using mice with Cre/LoxP conditional knockout alleles for CBP (CBPflox) and p300 (p300flox), which carry CD19Cre that initiates floxed gene recombination at the pro–B-cell stage. CD19Cre-mediated loss of CBP or p300 led to surprisingly modest deficits in B-cell numbers, whereas inactivation of both genes was not tolerated by peripheral B cells. There was a moderate decrease in B-cell receptor (BCR)–responsive gene expression in CBP or p300 homozygous null B cells, suggesting that CBP and p300 are essential for this signaling pathway that is crucial for B-cell homeostasis. These results indicate that individually CBP and p300 are partially limiting beyond the pro-B-cell stage and that other coactivators in B cells cannot replace their combined loss.

Description: Background: Langerhans cell histiocytosis (LCH) is a rare disorder characterized by clonal proliferation of Langerhans cells and significant infiltration of immune cells. Although the precise pathophysiology is to be elucidated, oncogenic BRAF-V600E mutation could be detected in LCH lesions from the majority of patients (Badalian-Very, et al. Blood. 2010). Furthermore, Berres ML et al. found that patients with active, high-risk LCH carried BRAF-V600E in circulating CD11c+/CD14+ cell fractions. In patients with various kinds of cancers, circulating cell-free DNA (cfDNA) in peripheral blood contains cancer-derived genomic DNA and has been applied to non-invasive diagnostic procedure, so called liquid biopsy. In the very recent report, BRAF-V600E was successfully detected on cfDNA from patients with colorectal cancer in 100% sensitivity and specificity (Thierry AR, et al. Nature Med. 2014). Then, in this study, we evaluated BRAF mutation on cfDNA as a potential biomarker of LCH using allele-specific quantitative polymerase chain reaction (ASQ-PCR). Methods: We cloned normal and mutant BRAF alleles that include exon 15 and neighboring sequences into pCR2.1 to make the standard curve. cfDNA was prepared from plasma of adult LCH patients and was subjected to genotyping BRAF alleles by ASQ-PCR, which was specifically designed for detection of BRAF-V600E with a 3'-phosphate-modified oligonucleotide blocker according to Thierry AR, et al. Mutant BRAF load was estimated from the standard curve in each assay and was expressed as the percentage of mutant alleles to total number of alleles. Results and Discussion: Plasma cfDNA was prepared from 8 adult patients with LCH listed in Table.1 as well as normal subjects including cancer-free patients. The mean quantity of recovered cfDNA in LCH vs normal was 316.5pg/ml (median, 290.4) vs 92.0pg/ml (median, 91.8). Three high-risk patients with active multiple lesions were positive for BRAF-V600E. In these patients, the mean ratio of mutant BRAF alleles to total was 3.25 % (median, 2.59 %). Next, we compared the sensitivity of ASQ-PCR of BRAF-V600E between cfDNA and cellular DNA in the same blood sample. Naturally, too much more DNA was recovered from mononuclear cells than plasma in the same blood volume, whereas the ratio of mutant to total alleles was more than 10-fold higher in cfDNA, suggesting that LCH-derived genomes are significantly enriched in cfDNA compared with cellular DNA, and that cfDNA is more adequate for liquid biopsy in LCH with BRAF-V600E. Then, in a BRAF-V600E-positive patient, we followed the mutant BRAF load during the course of initial chemotherapy. The ratio of mutant to total alleles was estimated as 1.00% prior to chemotherapy and not detectable after one course of chemotherapy consisting of vinblastine, prednisolone, methotrexate and 6-mercaptopurine. The validity of this ASQ-PCR data was confirmed by a series of routine imaging analysis performed at the same time. Taken together, ASQ-PCR of BRAF-V600E on cfDNA may contribute to planning of risk-based treatment as well as monitoring of treatment efficacy in LCH, especially in an active, high-risk group. A number of BRAF-targeted inhibitors have been approved or under clinical trial for various cancers with BRAF mutant, and one of those, vemurafenib is also active against LCH with BRAF-V600E (Haroche J, et al. Blood. 2013). Hereafter, the utility of BRAF-V600E on cfDNA should be validated in a large cohort of LCH patients. Conclusion: In spite of a very small cohort, we demonstrated the feasibility of BRAF-V600E on cfDNA as a biomarker of active, high-risk LCH. Disclosures: This work was supported by grants from "Japan LCH Study Group" (JLSG). Can't read 3B2 tag because stream don't exist.Tag: _LTable15 Disclosures Kobayashi: Japan LCH Study Group (JLSG): Research Funding. Fukuyama:Pfizer Japan Inc.: Employment. Tojo:Bristol-Myers Squibb.: Research Funding; Chugai Pharmaceutical Co Ltd.: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co Ltd.: Research Funding; Pfizer Japan Inc.: Research Funding; Celgene: Consultancy; Novartis Pharmaceuticals Japan.: Research Funding, Speakers Bureau.

Description: Somatic mutations of the ASXL1 gene are recurrently detected in age-related clonal hematopoiesis (CH). However, how ASXL1 mutations causes CH are not understood. Here, using knockin (KI) mice expressing a C-terminally truncated form of ASXL1-mutant (ASXL1-MT), we investigated the effect of ASXL1-MT on physiological aging in hematopoietic stem cells (HSCs). 　To examine the influence of ASXL1-MT on hematopoiesis, we bred the ASXL1-MT-KI mice with Vav-Cre transgenic mice. Young ASXL1-MT-KI mice (6-12 weeks) did not show significant changes in hematological parameters and differentiation status of peripheral blood. We observed the decreased frequency of hematopoietic stem and progenitor cells (HSPCs), including long-term HSCs (LT-HSCs). Competitive transplantation assays showed the reduced repopulation ability in ASXL1-MT-KI HSPCs. Thus, ASXL1-MT decreased the number and impaired the function of HSPCs in young mice. 　Next, we examined age-related changes in hematopoiesis caused by ASXL1-MT. Aged ASXL1-MT-KI mice displayed a myeloid-biased differentiation and hypocellular bone marrow, indicating the dysfunction of hematopoiesis. Interestingly, ASXL1-MT markedly increased the frequency of phenotypic LT-HSCs (pLT-HSCs) in aged mice (20-24 months). Competitive transplantation assays showed the impaired repopulation potential of pLT-HSCs from aged ASXL1-MT-KI mice. These data demonstrate that the increased pLT-HSCs in aged ASXL1-MT-KI mice are not functional HSCs with long-term repopulation potential. 　To elucidate how ASXL1-MT drives HSPC dysfunction, we conducted RNA-Seq analysis using HSPCs from young mice. This analysis revealed upregulation of mitochondrial genes in ASXL1-MT-KI HSPCs. In addition, MitoTracker staining, extracellular flux analyses and metabolome analyses demonstrated the enhanced mitochondrial metabolism in ASXL1-MT-KI HSPCs. We also found that the aberrantly elevated mitochondrial activity induced ROS overproduction and increased DNA damage, resulting in HSPC dysfunction. 　As a mechanism underlying the enhanced mitochondrial activity of ASXK1-MT-KI HSPCs, we revealed that ASXL1-MT activated the Akt/mTOR pathway in HSPCs. Treatment with an Akt inhibitor perifosine or an mTOR inhibitor rapamycin normalized the mitochondrial membrane potential and ROS levels in ASXL1-MT-KI HSPCs. Moreover, rapamycin treatment improved engraftment of ASXL1-MT-KI bone marrow cells after transplantation. These data indicate that the activated Akt/mTOR signaling leads to the enhanced mitochondrial activity, elevated ROS levels, and HSPC dysfunction in ASXL1-MT-KI mice. 　To assess the impact of the enhanced Akt/mTOR signaling on age-related changes in ASXL1-MT-KI mice, we administered rapamycin to aged ASXL1-MT-KI mice. Intriguingly, rapamycin treatment decreased the frequency of pLT-HSCs, and normalized the bone marrow cellularity in aged ASXL1-MT-KI mice. Cell cycle analysis revealed that pLT-HSCs in G0 phase were decreased in aged ASXL1-MT-KI mice, which was normalized by rapamycin treatment. These data demonstrate that the activated Akt/mTOR pathway provokes the aberrant expansion of pLT-HSCs in aged ASXL1-MT-KI mice. 　We next attempted to clarify the underlying mechanism of Akt activation in ASXL1-MT-KI mice. Immunoprecipitation experiments revealed that ASXL1-MT/BAP1 complex deubiquitinated AKT in 293T cells. To determine the role of endogenous Bap1 on Akt signaling, we assessed the effect of Bap1 deletion in murine bone marrow cells transformed by combined expression of SETBP1-D868N and ASXL1-MT (cSAM cells). A time course experiments showed Akt phosphorylation induced by IL-3 stimulation was attenuated and shortened in Bap1-depleted cSAM cells. These data suggest that ASXL1-MT/BAP1 complex deubiquitinate and stabilize phosphorylated Akt. 　In summary, we demonstrated that ASXL1-MT cooperated with BAP1 to promote AKT deubiquitination and activation. The activated Akt/mTOR pathway led to enhanced mitochondrial metabolism, elevated ROS levels and increased DNA damage. These molecular bases underlie the age-associated expansion of the pLT-HSC compartment. Our results underscore the possibility that CH can originate from a pLT-HSC with a limited repopulation potential. A pharmacological inhibition of the Akt/mTOR pathway could be a promising therapeutic intervention to individuals with CH harboring ASXL1 mutations. Disclosures No relevant conflicts of interest to declare.

Description: Additional sex combs-like 1 (ASXL1) mutations are frequently found in myeloid malignancies such as AML, MDS and MPNs. Our previous study on mutant Asxl1 expressing knock-in (Asxl1-MT KI) mice revealed that Asxl1-MT impaired normal hematopoiesis (Nagase R et al. J. Exp. Med. 2018). However, Asxl1-MT alone was insufficient to develop myeloid leukemia. Thus, additional factors are required for myeloid transformation in ASXL1-mutated cells. Retrovirus-mediated insertional mutagenesis approaches demonstrated susceptibility of Asxl1-MT KI cells to myeloid leukemia, and helped identifying Hematopoietically expressed homeobox (Hhex) gene as a common retrovirus integration site. Therefore, we here investigated the potential cooperation between ASXL1-MT and HHEX in myeloid leukemogenesis. We first assessed the effects of ASXL1-MT and HHEX on proliferation and differentiation of murine and human hematopoietic stem progenitor cells (HSPCs). Expression of HHEX enhanced proliferation and blocked differentiation of HSPCs expressing ASXL1-MT, while it showed only a modest effect in normal HSPCs. Some of the mice transplanted with cells expressing ASXL1-MT and HHEX developed myeloid leukemia. Expression of ASXL1-MT and HHEX dramatically promoted the growth of RUNX1-ETO-expressing Cord Blood (CB) cells in vitro by promoting cell cycle and inhibiting apoptosis. Moreover, ASXL1-MT and HHEX synergistically accelerated development of two distinct types of myeloid leukemia driven by RUNX1-ETO9a or FLT3-ITD in vivo. These data indicate that ASXL1-MT and HHEX cooperatively work in leukemic transformation of myeloid cells. Next, we evaluated the role of endogenous HHEX in ASXL1-MT-expressing leukemia cells. We here used two murine mutant ASXL1-expressing leukemia cells (cSAM cells: cells with combined expression of SETBP1 and ASXL1 mutations, cRAM cells: cells with combined expression of RUNX1 and ASXL1 mutations). Depletion of Hhex using CRISPR-Cas9 system profoundly attenuated the colony-forming ability and leukemogenicity of cSAM and cRAM cells. Similarly, depletion of HHEX attenuated the growth of human leukemia cell lines harboring an ASXL1 mutation including MEG-01 and Kasumi-1 by inducing apoptosis and differentiation. In contrast, the growth of ASXL1 wildtype cell lines such as THP-1 and U937 was unaffected by HHEX depletion. Thus, endogenous HHEX promotes survival of ASXL1-mutated leukemia cells. To elucidate the underlying molecular mechanisms, we performed RNA-seq using RUNX1-ETO expressing CB cells. Gene set enrichment analysis revealed that genes related to leukemia stem cells were more enriched in cells expressing both ASXL1-MT and HHEX than control cells. In combination with this RNA-seq data and ChIP-seq using 293T cells, we identified Myb and Etv5 as candidate genes upregulated by HHEX expression. We confirmed that ASXL1-MT and HHEX upregulated Myb and Etv5 in murine HSPCs by RT-qPCR and HHEX bound to promoter regions of MYB and ETV5 genes in HL-60 cells by ChIP-qPCR. Conversely, depletion of Hhex reduced expression of Myb and Etv5 in cSAM and cRAM cells. Luciferase reporter assay revealed that co-expression of ASXL1-MT and HHEX cooperatively enhanced promoter activity of MYB. In addition, preliminary expression analyses of primary cells showed that HHEX/ETV5 mRNA expression levels were significantly and MYB mRNA expression tended to be higher in primary AML samples including ASXL1 mutations compared to the ones of healthy controls (Beat AML). Finally, we assessed the role of MYB or ETV5 in leukemogenesis driven by ASXL1 mutations. Depletion of Myb or Etv5 reduced colony-forming activity in cSAM and cRAM cells by promoting apoptosis or differentiation, respectively. Furthermore, ectopic expression of MYB or ETV5 significantly reversed the reduced colony-forming activity of Hhex-depleted cSAM cells. Taken together, our study demonstrates that ASXL1-MT and HHEX are cooperative events promoting myeloid leukemogenesis through upregulation of MYB and ETV5. Disclosures Maciejewski: Novartis: Consultancy; Alexion: Consultancy.

Description: 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.