Search

Myelodysplastic syndromes (MDS) are hematopoietic stem and progenitor cell (HSPC) malignancies characterized by ineffective hematopoiesis and an increased risk of leukemia transformation. Epigenetic regulators are recurrently mutated in MDS, directly implicating epigenetic dysregulation in MDS pathogenesis. Here, we identified a tumor suppressor role of the acetyltransferase p300 in clinically relevant MDS models driven by mutations in the epigenetic regulators TET2, ASXL1, and SRSF2. The loss of p300 enhanced the proliferation and self-renewal capacity of Tet2-deficient HSPCs, resulting in an increased HSPC pool and leukemogenicity in primary and transplantation mouse models. Mechanistically, the loss of p300 in Tet2-deficient HSPCs altered enhancer accessibility and the expression of genes associated with differentiation, proliferation, and leukemia development. Particularly, p300 loss led to an increased expression of Myb, and the depletion of Myb attenuated the proliferation of HSPCs and improved the survival of leukemia-bearing mice. Additionally, we show that chemical inhibition of p300 acetyltransferase activity phenocopied Ep300 deletion in Tet2-deficient HSPCs, whereas activation of p300 activity with a small molecule impaired the self-renewal and leukemogenicity of Tet2-deficient cells. This suggests a potential therapeutic application of p300 activators in the treatment of MDS with TET2 inactivating mutations.

Hormesis describes a biphasic dose-response relationship generally characterized by a low-dose excitement and a high-dose inhibition. This phenomenon has been observed in the regulation of cell, organ, and organismic level. However, hormesis has not reported in oocytes. In this study, we observed, for the first time, hormetic responses of PIPP levels in oocytes by inhibitor of Akt1 or PKCδ. The expression of PIPP was detected by qPCR, immunofluorescent (IF), and Western Blot (WB). To observe the changes of PIPP levels, we used the inhibitors against pAkt1 (Ser473) or PKCδ, SH-6 or sotrastaurin with low and/or high-dose, treated GV oocytes and cultured for 4 h, respectively. The results showed that PIPP expression was significantly enhanced when oocytes were treated with SH-6 or sotrastaurin 10 μM, but decreased with SH-6 or sotrastaurin 100 μM. We also examined the changes of PIPP levels when GV oocytes were treated with exogenous PtdIns(3,4,5)P3 or LY294002 for 4 h. Our results showed that PIPP level was enhanced much higher under the treatment of 0.1 μM PtdIns(3,4,5)P3 than that of 1 μM PtdIns(3,4,5)P3, which is consistent with the changes of PIPP when oocytes were treated with inhibitors of pAkt1 (Ser473) or PKCδ. In addition, with PIPP siRNA, we detected that down-regulated PIPP may affect distributions of Akt, Cdc25, and pCdc2 (Tyr15). Taken together, these results show that the relationships between PIPP and Akt may follow the principle of hormesis and play a key role during release of diplotene arrest in mouse oocytes.

Melatonin, mostly released by the pineal gland, is a circadian rhythm-regulated and multifunctional hormone. Great advances in melatonin research have been made, including its role in rhythms of the sleep-wake cycle, retardation of ageing processes, as well as antioxidant or anti-inflammatory functions. Melatonin can scavenge free radicals such as reactive oxygen species (ROS), a key factor in reproductive functions. Melatonin plays an important role in oocyte maturation, fertilization and embryonic development as well. The concurrent use of melatonin increases the number of mature oocytes, the fertilization rate, and number of high-quality embryos, which improves the clinical outcome of assisted reproductive technology (ART). This review discusses the relationship between melatonin and human reproductive function, and potential clinical applications of melatonin in the field of reproductive medicine.

AML1-ETO (AE) is a fusion transcription factor, generated by the t(8;21) translocation, that functions as a leukemia promoting oncogene. Here, we demonstrate that TATA-Box Binding Protein Associated Factor 1 (TAF1) associates with K43 acetylated AE and this association plays a pivotal role in the proliferation of AE-expressing acute myeloid leukemia (AML) cells. ChIP-sequencing indicates significant overlap of the TAF1 and AE binding sites. Knockdown of TAF1 alters the association of AE with chromatin, affecting of the expression of genes that are activated or repressed by AE. Furthermore, TAF1 is required for leukemic cell self-renewal and its reduction promotes the differentiation and apoptosis of AE+ AML cells, thereby impairing AE driven leukemogenesis. Together, our findings reveal a role of TAF1 in leukemogenesis and identify TAF1 as a potential therapeutic target for AE-expressing leukemia., AML1-ETO is a fusion protein in which acetylation of lysine-43 is critical to leukemogenesis. Here, they show that TAF1 is required for AML1-ETO mediated gene expression such that it binds to acetylated AML1-ETO to facilitate the association of AML1-ETO with chromatin, and consequently, promotes leukemic self-renewal.

Autophagic lysosomal reformation, a key cellular process for maintaining lysosome homeostasis in elevated autophagy, so far has only been reported for cells under certain forms of starvation. For this reason, it is controversial that whether this phenomenon is starvation-specific and its importance in lysosomal regeneration at the late stage of autophagy is often challenged. Here we show that exogenous hydrogen peroxide (H2O2) induced lysosome depletion and recovery characteristic of autophagic lysosomal reformation, and we confirmed the occurrence of autophagic lysosomal reformation after H2O2 treatment by demonstrating Rab7 dissociation from autolysosomes, recruitment of Phosphatidylinositol 4-phosphate (PI4P) and clathrin to the surface of autolysosomes, and the existence of tubular "pro-lysosome" structures extending from autolysosomes. Similar to starvation, H2O2 caused an initial deactivation and a subsequent reactivation for mTOR, and mTOR reactivation was essential for ALR. Our results provided a first non-starvation example of autophagic lysosomal reformation and provide evidence for its importance for some autophagic processes other than that of starvation.

To mount an effective immune response against infectious pathogens or tissue injury, hematopoietic stem cells (HSCs) increase their proliferation and production of myeloid cells, including macrophages, which destroy the pathogens and repair the damaged tissue. Proper resolution of inflammation is essential to restore hematopoietic homeostasis, as unrestrained inflammation can result in life-threatening pathologies such as sepsis, autoimmune disorders and cancer. The molecular mechanisms that control the resolution of inflammation, and how these contribute to disease phenotypes, are poorly understood. BAF (SWI/SNF) complexes are ATPase dependent chromatin-remodeling complexes that play fundamental roles in transcription. BAF complexes use the energy of ATP to modulate the accessibility of transcription factors to DNA and thus, orchestrate the gene expression programs that control proliferation and cellular identity. Genes encoding for BAF subunits are frequently mutated in cancer and developmental disorders. In hematopoietic malignancies, loss-of-function mutations and low expression of specific BAF subunits have been reported in patients with anemia and bone marrow failure. Work from our lab previously demonstrated that the hematopoietic-specific BAF complex subunit Dpf2 cooperates with the transcription factor Runx1 to regulate myeloid differentiation. Based on these studies, we generated a hematopoietic-specific Dpf2 knock-out mouse model and found that mice lacking Dpf2 develop pancytopenia, anemia and an uncontrolled inflammatory response that results in early death. Dpf2-/- peripheral blood samples showed dysplastic features including increased number of polychromatophilic blood cells and Howell-Jolly bodies in erythrocytes. Histopathological analyses revealed the presence of fibrosis and prominent infiltration of histiocytes in multiple organs, including lungs, liver and spleen. Detailed chemical profiling of plasma showed increased levels of multiple pro-inflammatory cytokines, indicative of systemic inflammation. Flow cytometry analyses and Mass cytometry profiling further revealed an expansion of myeloid lineages, specifically monocytes and macrophages, concomitant with severe defects in lymphoid and erythroid differentiation. We also found that Dpf2-/-HSCs had increased serial re-plating capacity and a marked myeloid differentiation bias. To identify the transcription factor networks underlying these phenotypes, we performed RNAseq and ATACseq on control and Dpf2-/- HSCs. Gene Set Enrichment Analyses indicated that Dpf2-/- HSCs have extensive gene expression alterations in immune signaling and interferon response pathways, as well as leukocyte and erythroid differentiation. We also found that Dpf2 loss results in pronounced changes in expression and genomic accessibility of specific transcription factors that control macrophage differentiation and proliferation. Together, our mechanistic studies support a model whereby the absence of Dpf2 results in misregulation of the transcription factor networks that establish macrophage cell identity, leading to a marked increase in macrophage infiltrations and shortened survival of the mice. Treatment of the Dpf2-/-mice with clodronate-containing liposomes, which deplete macrophages from bone marrow and spleen, prolonged survival of the mice. Our work uncovers a novel role of Dpf2 in restraining inflammatory responses by controlling macrophage proliferation and function. Moreover, we propose that, in addition to their tumor suppressive roles in cancer, BAF complexes may have a central role in the prevention of immunopathologies. Disclosures Kadoch: Foghorn Therapeutics: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Other: Scientific founder, fiduciary board of directors member, scientific advisory board member, shareholder, and consultant for Foghorn Therapeutics (Cambridge, MA). . Vega:NCI: Research Funding.

The AML1-ETO fusion protein, generated by the t(8;21) chromosomal translocation, is causally involved in nearly 20% of acute myeloid leukemia (AML) cases. In leukemic cells, AML1-ETO resides in and functions through a stable protein complex, AML1-ETO–containing transcription factor complex (AETFC), that contains multiple transcription (co)factors. Among these AETFC components, HEB and E2A, two members of the ubiquitously expressed E proteins, directly interact with AML1-ETO, confer new DNA-binding capacity to AETFC, and are essential for leukemogenesis. However, the third E protein, E2-2, is specifically silenced in AML1-ETO–expressing leukemic cells, suggesting E2-2 as a negative factor of leukemogenesis. Indeed, ectopic expression of E2-2 selectively inhibits the growth of AML1-ETO–expressing leukemic cells, and this inhibition requires the bHLH DNA-binding domain. RNA-seq and ChIP-seq analyses reveal that, despite some overlap, the three E proteins differentially regulate many target genes. In particular, studies show that E2-2 both redistributes AETFC to, and activates, some genes associated with dendritic cell differentiation and represses MYC target genes. In AML patients, the expression of E2-2 is relatively lower in the t(8;21) subtype, and an E2-2 target gene, THPO , is identified as a potential predictor of relapse. In a mouse model of human t(8;21) leukemia, E2-2 suppression accelerates leukemogenesis. Taken together, these results reveal that, in contrast to HEB and E2A, which facilitate AML1-ETO–mediated leukemogenesis, E2-2 compromises the function of AETFC and negatively regulates leukemogenesis. The three E proteins thus define a heterogeneity of AETFC, which improves our understanding of the precise mechanism of leukemogenesis and assists development of diagnostic/therapeutic strategies.

Summary Chromatin-modifying enzymes, and specifically the protein arginine methyltransferases (PRMTs), have emerged as important targets in cancer. Here, we investigated the role of CARM1 in normal and malignant hematopoiesis. Using conditional knockout mice, we show that loss of CARM1 has little effect on normal hematopoiesis. Strikingly, knockout of Carm1 abrogates both the initiation and maintenance of acute myeloid leukemia (AML) driven by oncogenic transcription factors. We show that CARM1 knockdown impairs cell-cycle progression, promotes myeloid differentiation, and ultimately induces apoptosis. Finally, we utilize a selective, small-molecule inhibitor of CARM1 to validate the efficacy of CARM1 inhibition in leukemia cells in vitro and in vivo. Collectively, this work suggests that targeting CARM1 may be an effective therapeutic strategy for AML.

ASXL1 is frequently mutated in myeloid malignancies and is known to co-occur with other gene mutations. However, the molecular mechanisms underlying the leukemogenesis associated with ASXL1 and cooperating mutations remain to be elucidated. Here, we report that Asxl1 loss cooperated with haploinsufficiency of Nf1, a negative regulator of the RAS signaling pathway, to accelerate the development of myeloid leukemia in mice. Loss of Asxl1 and Nf1 in hematopoietic stem and progenitor cells resulted in a gain-of-function transcriptional activation of multiple pathways such as MYC, NRAS, and BRD4 that are critical for leukemogenesis. The hyperactive MYC and BRD9 transcription programs were correlated with elevated H3K4 trimethylation at the promoter regions of genes involving these pathways. Furthermore, pharmacological inhibition of both the MAPK pathway and BET bromodomain prevented leukemia initiation and inhibited disease progression in Asxl1(Δ/Δ) Nf1(Δ/Δ) mice. Concomitant mutations of ASXL1 and RAS pathway genes were associated with aggressive progression of myeloid malignancies in patients. This study sheds light on the effect of cooperation between epigenetic alterations and signaling pathways on accelerating the progression of myeloid malignancies and provides a rational therapeutic strategy for the treatment of myeloid malignancies with ASXL1 and RAS pathway gene mutations.

Epigenetic regulators play critical roles in normal hematopoiesis, and the activity of these enzymes is frequently altered in hematopoietic cancers. The major type II protein arginine methyltransferase PRMT5 catalyzes the formation of symmetric dimethyl arginine and has been implicated in various cellular processes, including pluripotency and tumorigenesis. Here, we generated Prmt5 conditional KO mice to evaluate the contribution of PRMT5 to adult hematopoiesis. Loss of PRMT5 triggered an initial but transient expansion of hematopoietic stem cells (HSCs); however, Prmt5 deletion resulted in a concurrent loss of hematopoietic progenitor cells (HPCs), leading to fatal BM aplasia. PRMT5-specific effects on hematopoiesis were cell intrinsic and depended on PRMT5 methyltransferase activity. We found that PRMT5-deficient hematopoietic stem and progenitor cells exhibited severely impaired cytokine signaling as well as upregulation of p53 and expression of its downstream targets. Together, our results demonstrate that PRMT5 plays distinct roles in the behavior of HSCs compared with HPCs and is essential for the maintenance of adult hematopoietic cells.

Myricitrin has many pharmacological effects, such as anti-inflammation, liver protection and anti-oxidation. However, its clinical application is limited by poor solubility and low oral bioavailability. The preparation of myricitrin-loaded proliposomes (MPs) was achieved via the combination of thin-film dispersion technique and freeze-drying method. The in vitro release of MPs compared with free myricitrin was measured in different dissolution media while the pharmacokinetic study was also conducted in rats. Moreover, the uric acid-lowering activity of MPs was investigated in the hyperuricemic rat model. The prepared myricitrin appeared to be spherical. Notably, compared with the free myricitrin, the cumulative release in vitro and in vivo oral bioavailability of MPs were markedly increased. Besides, the MPs could significantly lower the serum uric acid level as well as ameliorate liver and kidney damage in hyperuricemic rats compared with the model group. Therefore, the present work supports the fact that MPs improved the oral bioavailability of myricitrin for the prospect of clinical application.

Background Epigenetic dysregulation is a hallmark feature of myelodysplastic syndrome (MDS) as epigenetic regulator genes, such as ASXL1, TET2, and SRSF2, are highly mutated in MDS patients. Tet2 (ten-eleven translocation-2, a methylcytosine deoxygenase) mutations are found in ~20% of MDS patients; they are also seen in patients with acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPN). Loss of Tet2 in hematopoietic cells leads hematopoietic defects including enhanced hematopoietic stem cell (HSC) self-renewal, myeloid expansion and an increased propensity to develop MDS or AML. The lysine acetyltransferase (KAT) p300 (encoded by the EP300 gene) plays pivotal roles in essential cellular functions including proliferation, differentiation and signal transduction. However, the function of p300 in hematopoietic malignancies is clearly cell-context dependent, as we have shown that p300 functions as an oncogene in AML1-ETO-driven AML, and a tumor suppressor gene in NUP98-HOXD13-driven MDS. The EP300 and the related CREBBP genes are infrequently mutated in MDS patients. Methods To investigate the role of p300 in MDS driven by lack of Tet2, we generated a mouse model carrying a hematopoietic-specific EP300 conditional knockout on a Tet2-null background and characterized the effect of p300 loss on HSC function and the pathogenesis of MDS and AML. We also explored the therapeutic potential of manipulating p300 KAT activity to affect the course of MDS driven by Tet2 loss. Results Compared to the Tet2-/- mice, the EP300Δ/ΔTet2-/- (DKO) mice had shortened survival and an accelerated onset of AML, with increased blasts in the bone marrow and peripheral blood, anemia, splenomegaly, and universal presence of granulocytic sarcomas, demonstrating that depletion of p300 enhances the leukemogenicity of Tet2-/- HSC. The DKO mice showed enhanced hematopoietic stem/progenitor cell (HSPC, defined as Lin-Sca1+Kit+ cell) proliferation and an increased HSPC pool only 2 weeks after p300 deletion, far in advance of the development of any disease manifestations. In contrast, deletion of p300 in wild type (wt) mice affected neither HSPC proliferation, nor pool size. To further define the mechanisms by which these two genetic events cooperate to drive the development of MDS/AML, we assessed the DNA methylation status and gene expression signatures of HSPCs isolated from wt, Tet2-/- and DKO mice. As expected, loss of Tet2 leads to a genome-wide decrease in 5-hmC, whereas loss of p300 does not change 5-hmC distribution across the genome. Interestingly, DKO HSPCs showed local gains of 5-hmC at a variety of genes (compared to Tet2-null HSPCs), including at the c-Myb gene, which is a known oncogenic driver of AML. Gene Set Enrichment Analysis (GSEA) revealed that p300 loss in Tet2-/- HSPCs interferes with leukocyte differentiation, enhances proliferation and inhibits apoptosis. ChIP-X Enrichment Analysis (ChEA) further also indicated that among the differentially expressed genes, between DKO HSPCs and Tet2-null HSPCs, are potential Myb target genes. Given this data and the RNA-Seq and Q-PCR results that showed elevated levels of Myb mRNA in the DKO HSPCs compared to the Tet2-/- HSPCs, we examined the biological effects of knocking down (KD) Myb. Myb KD reduced the colony-forming capacity of the DKO HSPCs, but not the Tet2 -/- HSPCs, identifying its potential role in mediating the effect of p300 on MDS prone HSPCs. These results indicate that loss of p300 in Tet2-null HSPCs enhances their leukemogenicity through elevated Myb expression and activity. To determine whether increasing p300 KAT activity could have an opposite effect on MDS cells, we utilized I-CBP112, which binds p300 and increases its KAT activity and ability to stimulate H3K18 acetylation. Exposure to I-CBP112 did in fact eliminate the indefinite self-renewal capacity of Tet2-/- HSPCs, as measured by serial replating assays, further highlighting the importance of p300 KAT activity in regulating the behavior of Tet2-/- HSPCs. Additional in vivo studies of the effects of I-CBP112 on the course of MDS/AML driven by the absence of Tet2 are ongoing. Conclusions This work clearly indicates that the EP300 gene can play a tumor suppressor role in MDS/AML driven by loss of Tet2- by repressing Myb expression and activity. It also suggests that the enhancement of p300's KAT activity by use of small molecules, could be an effective therapeutic strategy for MDS/AML. Disclosures Cole: Abbvie: Consultancy; Acylin Therapeutics: Equity Ownership.

AML1-ETO (AE), a fusion oncoprotein generated by t(8;21), can trigger acute myeloid leukemia (AML) in collaboration with mutations including c-Kit, ASXL1/2, FLT3, N-RAS, and K-RAS. Caspase-3, a key executor among its family, plays multiple roles in cellular processes, including hematopoietic development and leukemia progression. Caspase-3 was revealed to directly cleave AE in vitro, suggesting that AE may accumulate in a Caspase-3-compromised background and thereby accelerate leukemogenesis. Therefore, we developed a Caspase-3 knockout genetic mouse model of AML and found that loss of Caspase-3 actually delayed AML1-ETO9a (AE9a)-driven leukemogenesis, indicating that Caspase-3 may play distinct roles in the initiation and/or progression of AML. We report here that loss of Caspase-3 triggers a conserved, adaptive mechanism, namely autophagy (or macroautophagy), which acts to limit AE9a-driven leukemia. Furthermore, we identify ULK1 as a novel substrate of Caspase-3 and show that upregulation of ULK1 drives autophagy initiation in leukemia cells and that inhibition of ULK1 can rescue the phenotype induced by Caspase-3 deletion in vitro and in vivo. Collectively, these data highlight Caspase-3 as an important regulator of autophagy in AML and demonstrate that the balance and selectivity between its substrates can dictate the pace of disease.

ASXL2 is frequently mutated in acute myeloid leukaemia patients with t(8;21). However, the roles of ASXL2 in normal haematopoiesis and the pathogenesis of myeloid malignancies remain unknown. Here we show that deletion of Asxl2 in mice leads to the development of myelodysplastic syndrome (MDS)-like disease. Asxl2−/− mice have an increased bone marrow (BM) long-term haematopoietic stem cells (HSCs) and granulocyte–macrophage progenitors compared with wild-type controls. Recipients transplanted with Asxl2−/− and Asxl2+/− BM cells have shortened lifespan due to the development of MDS-like disease or myeloid leukaemia. Paired daughter cell assays demonstrate that Asxl2 loss enhances the self-renewal of HSCs. Deletion of Asxl2 alters the expression of genes critical for HSC self-renewal, differentiation and apoptosis in Lin−cKit+ cells. The altered gene expression is associated with dysregulated H3K27ac and H3K4me1/2. Our study demonstrates that ASXL2 functions as a tumour suppressor to maintain normal HSC function., ASXL2 mutations are mostly found in a subset of leukemia patients with certain genetic aberrations; however the role of this protein in normal hematopoiesis and related malignancies is still unclear. Here the authors use a knock-out mouse model to uncover the role of Asxl2 in hematopoiesis and leukemogenesis.

Herein, a kind of novel monocomponent hydrophilic and paramagnetic manganese(II) oxide nanocrystal is prepared in polar solution by a one-pot microwave-assisted synthesis. This kind of nanocrystal can be taken up effi ciently to serve as an excellent T 1 magnetic resonance imaging (MRI) contrast agent with an enhanced r 1 value of 0.81 mM − 1 s − 1 . The key to the success of this method is that no additional capping agents are required for coating onto the surface via ligand exchange, facilitating research of their intrinsic biological activities. Furthermore, multiple lines of convincing evidence are presented to prove that MnO nanocrystals (NCs) elicit p53-activationindependent and authentic functional autophagy via inducing autophagosome formation. Notably, there are very few reports so far of the autophagy phenomenon induced by magnetic nanocrystals. Moreover, these results offer an indication for cancer therapy that MnO NCs combined with doxorubicin at a nontoxic concentration can have a defi nite synergistic effect, which is mediated through the genuine autophagy induction, on killing cancer cells in vitro and in vivo.

Cysteamine (CS) has many biomedical and clinical applications because of its excellent water solubility, low cytotoxicity and good biocompatibility. A previous study by Brawer et al. reported the occurrence of many Gomori inclusion bodies in CS-treated astrocytes, which would suggest the induction of autophagy. Here we provided a comprehensive line of evidence demonstrating that CS caused autophagosome accumulation in cancer cells. CS exerted a biphasic effect on the autophagy process, increasing the formation of autophagosomes in the early phase and blocking the autophagic degradation in a later phase. Furthermore, we showed that CS sensitized doxorubicin-elicited chemotherapeutic killing in HeLa, B16 melanoma and doxorubicin-resistant MCF-7 cells and also enhanced chemotherapeutic efficacy of doxorubicin in a mouse melanoma model. Finally, we demonstrated that the chemosensitizing effect of CS was at least partly dependent on its ability to modulate autophagy. Our results revealed a novel biological function for CS in enhancing the chemotherapeutic effect of doxorubicin through autophagy modulation and pointed to the potential use of CS in adjunct cancer chemotherapy.

Small molecule protein arginine methyltransferase inhibitors (PRMTi) are being actively pursued for the treatment of a variety of cancers; however, the mechanisms of response to PRMTi remain poorly understood. CARM1, also known as PRMT4, is significantly overexpressed in AML, as well as many solid tumors, and regulates myeloid differentiation. We have shown the dependency of AML cells, but not normal blood cells, on CARM1 activity, based on CARM1 knockout, CARM1 knockdown, and chemical inhibition (Greenblatt et al. Cancer Cell 2018). These experiments showed that CARM1 regulates essential processes in leukemia cells, and is critical for leukemic transformation. Although several small molecule inhibitors of CARM1 have been reported recently, many display a lack of selectivity for CARM1 or fail to produce a biological response. The recent discovery of potent and selective CARM1 inhibitors (Drew et al., 2017), has made it possible to investigate the implications of pharmacological inhibition of CARM1 in vitro and in vivo. In vitro, a selective CARM1 inhibitor, EPZ025654, reduced the methylation of a CARM1 substrate, BAF155, in a time and concentration-dependent manner, while the specific histone targets of CARM1 remained unchanged. Translocation (8;21) AML samples in the Eastern Cooperative Oncology Group cohort, have significantly higher CARM1 expression compared to normal CD34+ controls. This led us to hypothesize that CARM1 is a direct target of the AML1-ETO fusion protein. Therefore, we assessed whether EPZ025654 could inhibit AML1-ETO driven gene expression. AML1-ETO specific target genes showed significant changes in expression following EPZ025654 treatment. AML1-ETO positive patient samples also displayed decreased colony formation in methylcellulose and increased myeloid differentiation in response to CARM1 inhibition. We next evaluated EZM2302, a compound structurally related to EPZ025654, that is highly orally bioavailable and is well tolerated in mice (Drew et al., 2017). We generated AE9a-GFP primary transplantation mice and treated them with 100 mg/kg of EZM2302 or vehicle twice-daily (BID). The inhibitor treated mice showed significantly improved survival as well as fewer GFP+ cells in the peripheral blood over time. GFP+ AE9a bone marrow cells also showed decreased colony formation in vitro and induced macrophage differentiation in methylcellulose. GFP+ cells were isolated by FACS and submitted for RNA-sequencing. Flow cytometry analysis post-treatment revealed a significant downregulation of c-Kit and increased differentiation of hematopoietic stem and progenitor cells. Resistance to epigenetic targeted therapeutics has been observed, often through the induction of kinase signaling pathways. Therefore, we explored synergistic combinations with CARM1 inhibition using RNA-sequencing and proteomics analysis in leukemia cell lines. We used L1000 profiling (Subramanian et al., 2017) to simultaneously profile the transcriptional response of 18 AML cell line and CD34+ cells after 6 days of treatment. The AML1-ETO positive cell lines exhibited an IC50 in the 0.4-3 μM range, while CD34+ cells and several AML cell lines appeared to be resistant to CARM1 inhibition. While gene expression changes resulting from alterations in RNA stability were observed, the most significant differences between sensitive and resistant cell lines were genes associated with the regulation of cell cycle progression. Gene expression changes were evaluated over time in an AML1-ETO positive cell line, SKNO-1. SKNO-1 cell lines showed an upregulation of a gene expression signature associated with PI3K/AKT/mTOR signaling, with the most significant gene expression changes occurring 7-14 days post treatment. We simultaneously profiled these cells using multiplexed kinase inhibitor beads (MIBs) and quantitative mass spectrometry (MS) to compare kinase expression and activity in response to CARM1 inhibition over time. A comparison of this response to chemical perturbation signatures in the L1000 database, identified several chemical inhibitors of the PI3K/AKT/mTOR axis that could reverse the gene expression changes induced by CARM1 inhibition. This finding elucidated a response mechanism for CARM inhibition and a synergistic therapeutic strategy that has the potential to improve CARM1 directed therapy. Disclosures No relevant conflicts of interest to declare.

Inhibitor of DNA binding 1 (Id1) functions as an E protein inhibitor, and overexpression of Id1 is seen in acute myeloid leukemia (AML) patients. To define the effects of Id1 on leukemogenesis, we expressed MLL-AF9 in fetal liver (FL) cells or bone marrow (BM) cells isolated from wild-type, Id1(-/-), p21(-/-), or Id1(-/-)p21(-/-) mice, and transplanted them into syngeneic recipient mice. We found that although mice receiving MLL-AF9-transduced FL or BM cells develop AML, loss of Id1 significantly prolonged the median survival of mice receiving FL cells but accelerated leukemogenesis in recipients of BM cells. Deletion of Cdkn1a (p21), an Id1 target gene, can rescue the effect of Id1 loss in both models, suggesting that Cdkn1a is a critical target of Id1 in leukemogenesis. It has been suggested that the FL transplant model mimics human fetal-origin (infant) MLL fusion protein (FP)-driven leukemia, whereas the BM transplantation model resembles postnatal MLL leukemia; in fact, the analysis of clinical samples from patients with MLL-FP(+) leukemia showed that Id1 expression is elevated in the former and reduced in the latter type of MLL-FP(+) AML. Our findings suggest that Id1 could be a potential therapeutic target for infant MLL-AF9-driven leukemia.

Histone or non-histone protein acetylation plays important roles in all kinds of cellular events, including the normal and abnormal development of blood cells, through changing the epigenetic status of chromatin and regulating non-histone protein’s function. Histone acetyltransferases (HATs), which are the enzymes responsible for the histone or non-histone protein acetylation, contain p300/CBP, MYST and GNAT family etc. HATs are not only the protein modifiers and epigenetic factors, but also the critical regulators of cell development and cancerogenesis. Here we will review the function of HATs such as p300/CBP, Tip60, MOZ/MORF and GCN5/PCAF in the normal hematopoiesis and the pathogenesis of hematological malignancies. The inhibitors that have been developed to target HATs will also be reviewed here. Understanding the roles of HATs in normal/malignant hematopoiesis and the underlying mechanism will provide the potential therapeutic targets for the hematological malignancies.

Transcriptional regulators are recurrently altered through translocations, deletions, or aberrant expression in acute myeloid leukemia (AML). Although critically important in leukemogenesis, the underlying pathogenetic mechanisms they trigger remain largely unknown. Here, we identified that Id1 (inhibitor of DNA binding 1) plays a pivotal role in acute myeloid leukemogenesis. Using genetically modified mice, we found that loss of Id1 inhibited t(8;21) leukemia initiation and progression in vivo by abrogating protein kinase B (AKT)1 activation, and that Id1 interacted with AKT1 through its C terminus. An Id1 inhibitor impaired the in vitro growth of AML cells and, when combined with an AKT inhibitor, triggered even greater apoptosis and growth inhibition, whereas normal hematopoietic stem/progenitor cells were largely spared. We then performed in vivo experiments and found that the Id1 inhibitor significantly prolonged the survival of t(8;21)(+) leukemic mice, whereas overexpression of activated AKT1 promoted leukemogenesis. Thus, our results establish Id1/Akt1 signaling as a potential therapeutic target in t(8;21) leukemia.

Chromatin modifying enzymes, and specifically the protein arginine methyltransferases (PRMTs) have emerged as important targets in cancer. PRMT4, also known as CARM1, is overexpressed in a number of cancers, including breast, prostate, pancreatic, and lung cancer. Our lab reported the overexpression of PRMT4 in the context of acute myeloid leukemia (AML), showing that more than 70% of cytogenetically normal AML patients have up-regulation of PRMT4. Here, we investigated the role of PRMT4 in normal hematopoiesis and leukemia development. In order to study the role of PRMT4 in normal hematopoiesis, Prmt4-floxed mice were crossed with Vav1-cre mice purchased from the Jackson Laboratory. Inducible Prmt4 conditional KO mice were generated by crossing Prmt4-floxed mice with Mx1-Cre mice and Prmt4 gene excision was induced by poly(I:C). Using this hematopoietic specific knockout system, we show that loss of PRMT4 has little effect on normal hematopoiesis, but promotes the differentiation of hematopoietic stem and progenitor cells. Next we evaluated the role of PRMT4 in leukemia development using leukemia transplantation models driven by fusion oncoproteins. Strikingly, the knockout of PRMT4 completely abrogates leukemia initiation in fetal liver transplantation models driven by the AML1-ETO or MLL-AF9 fusion proteins. We further characterized the mechanism for the leukemia-specific dependence on PRMT4 using leukemia cell lines and found that knockdown of PRMT4 impairs cell cycle progression, decreases proliferation, and induces rapid apoptosis. To examine PRMT4 dependent changes in gene expression in a leukemia system, we used lentiviral vectors that express RFP and shRNAs directed against PRMT4. We knocked down PRMT4 in four leukemia cell lines or normal human cord-blood derived CD34+ cells. Gene set enrichment analysis showed that all four leukemia cell lines with knockdown of PRMT4 significantly down-regulated E2F target genes compared to the scrambled control. Chromatin immunoprecipitation analysis (ChIP) confirmed the presence of PRMT4 and H3R17 dimethylation at the promoter regions of E2F1 targets. The PRMT4 conditional knockout mice and PRMT4 knockdown experiments both suggest that the loss of PRMT4 protein has a selective effect on leukemia cells compared to normal hematopoietic stem and progenitor cells. Collectively, this work suggests that targeting PRMT4 through chemical inhibition may be an effective therapeutic strategy for AML and other cancers with up-regulation of PRMT4. Citation Format: Sarah M. Greenblatt, Pierre-Jacques J. Hamard, Takashi Asai, Na Man, Concepcion Martinez-Caja, Fan Liu, Stephen Nimer. Identification of CARM1/PRMT4 as a novel therapeutic target for AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3340. doi:10.1158/1538-7445.AM2017-3340

The possible adverse effects of engineered nanomaterials on human health raise increasing concern as our research on nanosafety intensifies. Upon entry into a human body, whether intended for a theranostic purpose or through unintended exposure, nanomaterials tend to accumulate in the liver, leading to hepatic damage. A variety of nanoparticles, including rare earth upconversion nanoparticles (UCNs), have been reported to elicit hepatotoxicity, in most cases through inducing immune response or activating reactive oxygen species. Many of these nanoparticles also induce autophagy, and autophagy inhibition has been shown to decrease UCN-induced liver damage. Herein, using UCNs as a model engineered nanomaterial, this study uncovers a critical role for Kupffer cells in nanomaterial-induced liver toxicity, as depletion of Kupffer cells significantly exacerbates UCN-induced liver injury. Furthermore, UCN-induced prodeath autophagy in Kupffer cells, and inhibition of autophagy with 3-MA, a well-established chemical inhibitor of autophagy, enhances Kupffer cell survival and further abrogates UCN-induced liver toxicity. The results reveal the critical importance of Kupffer cell autophagy for nanoparticle-induced liver damage, and inhibition of autophagy may constitute a novel strategy for abrogating nanomaterial-elicited liver toxicity.

Background: Rearrangement between chromosome 8 and 21 is the most frequently observed chromosomal translocation. In AML patients, it leads to the formation of a novel fusion protein AML1-ETO. AML1-ETO functions as a transcriptional regulator to suppress myeloid differentiation and to promote self-renewal of hematopoiesis stem cells, which are of importance in leukemia development. However, AML1-ETO has no enzymatic function, thus targeting AML1-ETO directly is technically difficult. TAFII250, a largest subunit of the transcription factor IID complex (TFIID) serves to bring other components of basal transcription machinery to the preinitiation complex during the transcription initiation. And TAFII250 also plays a crucial role in the expression of genes involved in cell cycle and apoptosis. Yet despite the importance of these processes, there is no direct evidence implicating TAFII250 in cancer development. We have reported that acetylation of AML1-ETO on lysine 43 is critical for AML1-ETO mediated leukemogenesis and used a peptide pulldown strategy to identify proteins that preferring interact with acetylated AML1-ETO and identified TAFII250. Here, we are going to report the functions of TAFII250 in AML1-ETO induced leukemogenesis. Methods: We used Kasumi-1 and SKNO-1 cell lines derived from t(8; 21)AML patients, and a mouse AML1-ETO exon 9a (AE9a) expressing cell line developed from bone marrow cells of mice injected with bone marrow cells infected with AE9a to perform in vitro and in vivo studies. Results: We demonstrate that TAFII250 associates with AML1-ETO in AML cells through its bromodomain recognizing acetylated lysine 43 on AML1-ETO. The knockdown of TAFII250 completely abolished the proliferation of AML1-ETO expressing cells and has little influence on cell growth of non AML1-ETO expressing cells K562 and CD34+ cells. In addition, when we examined cleaved caspase 3 and PARP by western and measured annexin-V through flow cytometry, we found that the deficiency of TAFII250 induces apoptosis in AML1-ETO expressing cells. Further, the co-immunoprecipitation assay revealed that the deficiency of TAFII250 blocks the binding of AML1-ETO to CBFb (core binding factor beta subunit) and the recruitment of AML1-ETO at the promoter regions of a subset of its target genes. Consequently, loss of TAFII250 interferes with the expression of these genes. The depletion of TAFII250 also has negative effect on the self-renewal of leukemic cells, promoting their differentiation. Most importantly, the loss of TAFII250 severely impairs leukemia development in AE9a expressing cells. Conclusions: Together, these results reveal an essential role of TAFII250 in AML1-ETO mediated leukemogenesis and imply that TAFII250 might be a therapeutic target for AML1-ETO expressing AMLs. Disclosures No relevant conflicts of interest to declare.

Background AML1-ETO (AE), a oncogenic protein generated by the t(8;21) translocation, causes acute myeloid leukemia (AML) in collaboration with other secondary events. The leukemogenicity of AE has been evaluated in multiple mouse models, such as expression of AE in Cdkn1a-null Hematopoietic stem cell (HSCs) and expression of AML1- ETO9a (AE9a), an alternatively spliced variant of AE, in WT HSCs. Both lead to the development of fully penetrant AML. Caspase-3 plays multiple roles in hematopoietic development and leukemia progression and treatment by affecting proliferation, self-renewal and differentiation. It has been shown that uncleaved caspase-3 levels are higher in the peripheral blood cells of AML patients compared to normal individuals, which suggests that the caspase pathway is dysregulated in AML. We and others have shown that Caspase-3 directly cleave AE in vitro, suggesting that AE may accumulate in a Caspase-3 compromised background and accelerate leukemogenesis. Methods We developed a Caspase-3 knockout genetic mouse model of AML based on fetal liver cell transplantation. In brief, fetal liver cells from WT or Caspase3-/- mice were transduced to express AE9a in vitroand 100,000 AE9a+ transduced cells were transplanted into lethally irradiated recipient mice by tail-vein injection. Results We found loss of Caspase-3 impaired leukemia stem cell (LSC) self-renewal and delayed AE9a-driven leukemogenesis, indicating that Caspase-3 may play distinct roles in the initiation or progression of AML. Moreover, we identified a new substrate of Caspase-3, ULK1, by in vitro cleavage assays and site-directed mutagenesis. ULK1 (serine/threonine UNC-51-like kinase) is the homology of Atg1 (the first autophagy related gene found in 1997) in mammalian cells, which is a direct target of mTOR and is responsible for initiation of the autophagic activity by forming a complex with mAtg13, FIP200 and Atg101. The induction of autophagy caused by upregulation of ULK1 in AE/AE9a-expressing Caspase-3-/- fetal liver cells acted to limit the leukemogenicity of AE9a in vivo. Inhibition of ULK1 by inhibitor or shRNAs could rescue the self-renewal capability induced by Caspase-3 deletion in serial replating assays. Unexpectedly, when we expressed AE/AE9a in fetal liver cells from WT and Caspase-3-/- mice, the protein levels were comparable suggesting the basal level of Caspase-3 didn't affect the expressing of AE/AE9a in fetal liver cells. Conclusion Autophagy may play a general role in the development and treatment of leukemia. In human AML, blasts display reduced expression of autophagy-related genes and decreased autophagic flux, indicating that low autophagy activity provides a general advantage for leukemia development. Beside this, a number of chemotherapy drugs have been reported to be able to induce leukemia cell death via activation of autophagy suggesting that autophagy plays critical roles in the leukemia treatment. Our study reveals that Caspase-3 regulates autophagy through its direct cleavage of ULK1 and this interaction dictates the pace of AE-driven leukemogenesis. Targeting this pathway may have therapeutic benefit for AML treatment. Disclosures No relevant conflicts of interest to declare.

Omi, also known as high temperature requirement factor A2 (HtrA2), is a serine protease that was originally identified as a proapoptotic protein. Like Smac/Diablo, it antagonizes inhibitor of apoptosis proteins when released into the cytosol on apoptotic stimulation. Loss of its protease activity in mnd2 (motor neuron degeneration 2) mice is associated with neurodegeneration. However, the detailed mechanisms by which Omi regulates the pathogenesis of neurodegenerative disease remain largely unknown. We report here that Omi participates in the pivotal cellular degradation process known as autophagy. It activates autophagy through digestion of Hax-1, a Bcl-2 family-related protein that represses autophagy in a Beclin-1 (mammalian homologue of yeast ATG6)-dependent pathway. Moreover, Omi-induced autophagy facilitates the degradation of neurodegenerative proteins such as pathogenic A53T α-synuclein and truncated polyglutamine-expanded huntingtin, as well as the endogenous autophagy substrate p62. Knockdown of Omi decreases the basal level of autophagy and increases the level of the above target proteins. Furthermore, S276C Omi, the protease-defective mutant found in mnd2 mice, fails to regulate autophagy. Increased autophagy substrates and the formation of aggregate structures are observed in the brains of mnd2 mice. These results identify Omi as a novel regulator of autophagy and suggest that Omi might be important in the cellular quality control of proteins involved in neurodegenerative diseases.