Design of Inhibitors That Target the Menin-Mixed-Lineage Leukemia Interaction.

The prognosis of mixed-lineage leukemia (MLL) has remained a significant health concern, especially for infants. The minimal treatments available for this aggressive type of leukemia has been an ongoing problem. Chromosomal translocations of the KMT2A gene are known as MLL, which expresses MLL fusion proteins. A protein called menin is an important oncogenic cofactor for these MLL fusion proteins, thus providing a new avenue for treatments against this subset of acute leukemias. In this study, we report results using the structure-based drug design (SBDD) approach to discover potential novel MLL-mediated leukemia inhibitors from natural products against menin. The three-dimensional (3D) protein model was derived from Protein Databank (Protein ID: 4GQ4), and EasyModeller 4.0 and I-TASSER were used to fix missing residues during rebuilding. Out of the ten protein models generated (five from EasyModeller and I-TASSER each), one model was selected. The selected model demonstrated the most reasonable quality and had 75.5% of residues in the most favored regions, 18.3% of residues in additionally allowed regions, 3.3% of residues in generously allowed regions, and 2.9% of residues in disallowed regions. A ligand library containing 25,131 ligands from a Chinese database was virtually screened using AutoDock Vina, in addition to three known menin inhibitors. The top 10 compounds including ZINC000103526876, ZINC000095913861, ZINC000095912705, ZINC000085530497, ZINC000095912718, ZINC000070451048, ZINC000085530488, ZINC000095912706, ZINC000103580868, and ZINC000103584057 had binding energies of -11.0, -10.7, -10.6, -10.2, -10.2, -9.9, -9.9, -9.9, -9.9, and -9.9 kcal/mol, respectively. To confirm the stability of the menin-ligand complexes and the binding mechanisms, molecular dynamics simulations including molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) computations were performed. The amino acid residues that were found to be potentially crucial in ligand binding included Phe243, Met283, Cys246, Tyr281, Ala247, Ser160, Asn287, Asp185, Ser183, Tyr328, Asn249, His186, Leu182, Ile248, and Pro250. MI-2-2 and PubChem CIDs 71777742 and 36294 were shown to possess anti-menin properties; thus, this justifies a need to experimentally determine the activity of the identified compounds. The compounds identified herein were found to have good pharmacological profiles and had negligible toxicity. Additionally, these compounds were predicted as antileukemic, antineoplastic, chemopreventive, and apoptotic agents. The 10 natural compounds can be further explored as potential novel agents for the effective treatment of MLL-mediated leukemia.

LncRNA HOTTIP regulates TLR4 promoter methylation by recruiting H3K4 methyltransferase MLL1 to affect apoptosis and inflammatory response of fibroblast-like synoviocyte in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease, and the role of HOXA transcript at the distal tip (HOTTIP) in its pathogenesis remains underexplored. This study investigates the mechanism by which HOTTIP influences apoptosis and the inflammatory response of fibroblast-like synoviocytes (FLS). An RA mouse model was established, and clinical scores were analyzed. Pathological changes in synovial tissues, bone mineral density (BMD) of the paws, serum tartrate-resistant acid phosphatase (TRAP) activity, and TNF-α and IL-1ß levels were assessed. FLS were transfected, and cell proliferation and apoptosis were examined. The RNA-pull-down assay determined HOTTIP's interaction with mixed-lineage leukemia 1 (MLL1), while RNA immunoprecipitation assay measured HOTTIP expression pulled down by MLL1. The levels of MLL1 and toll-like receptor 4 (TLR4) after MLL1 overexpression based on HOTTIP silencing were determined. Chromatin immunoprecipitation (ChIP) was performed with H3K4me3 as an antibody, followed by the evaluation of TLR4 expression. HOTTIP expression was elevated in RA mouse synovial tissues. Inhibition of HOTTIP led to reduced clinical scores, inflammatory infiltration, synovial hyperplasia, TRAP activity, and TNF-α and IL-1ß levels, along with increased BMD. In vitro Interference with HOTTIP suppressed RA-FLS apoptosis and inflammation. HOTTIP upregulated TLR4 expression by recruiting MLL1 to facilitate TLR4 promoter methylation. MLL1 overexpression reversed HOTTIP silencing-mediated repression of RA-FLS apoptosis. Activation of H3K4 methylation counteracted HOTTIP knockout, ameliorating the inflammatory response. HOTTIP regulates TLR4 expression by recruiting MLL1, leading to TLR4 promoter methylation, thereby suppressing RA-FLS proliferation and inducing cell apoptosis and inflammatory response in RA.

Differentiation of Acute Leukemia Cells Including Cells with MLL-AF4 Rearrangements Induced by Jiyuan Oridonin A.

BACKGROUND: Chromosomal rearrangements involving the Mixed lineage leukemia (MLL) gene are observed in acute leukemia (AL) patients, which have poor prognosis, especially in infants. Hence, there is still a challenge to develop other effective agents to treat AL with MLL rearrangements (MLLr). MLL has been shown to rearrange with partner genes, of which the most frequently observed are AF4 and AF9. Moreover, AL is characterized by a differentiation blockage resulting in the accumulation of immature cells. An ent-kaurene diterpenoid compound, Jiyuan Oridonin A (JOA), has been shown to reduce the viability of AML cells by differentiation. METHODS: We aimed to evaluate the effect of JOA on the growth and differentiation of AL cells (SEM, JURKAT and MV4-11) including cells with MLLr-AF4 by cell proliferation assay, colony formation assay, cell cycle analysis, cell apoptosis analysis, measurement of cell surface antigens, cell morphology, mRNA-sequencing analysis, quantitative Real-time PCR and Western blotting analysis. RESULTS: Our findings demonstrated that the proliferation of AL cells including cells with MLLr-AF4 was significantly suppressed by JOA, which induced cell differentiation followed by G0/G1 cell cycle withdrawal. Moreover, JOA-mediated cell differentiation was likely due to activation of G-CSFR in MV4-11 cells. CONCLUSION: Our results suggest that JOA may be considered a promising anti-leukemia compound to develop to surmount the differentiation block in AL patients.

Myeloid sarcoma as the only manifestation in a rare mixed lineage leukemia-fusion-driven acute myeloid leukemia: A case report.

BACKGROUND: The mixed lineage leukemia (MLL)-eleven-nineteen lysine-rich leukemia (ELL) fusion gene is a rare occurrence among the various MLL fusion genes. We present the first case in which myeloid sarcoma (MS) was the only manifestation of adult MLL-ELL-positive acute myeloid leukemia (AML). CASE SUMMARY: We report a case of a 33-year-old male patient who was admitted in June 2022 with a right occipital area mass measuring approximately 7 cm × 8 cm. Blood work was normal. The patient underwent right occipital giant subscalp mass excision and incisional flap grafting. Immunohistochemistry was positive for myeloperoxidase, CD43 and CD45 and negative for CD3, CD20, CD34, and CD56. The bone marrow aspirate showed hypercellularity with 20% myeloblasts. Flow cytometry showed that myeloblasts accounted for 27.21% of the nucleated cells, which expressed CD33, CD38, and CD117. The karyotype was 46, XY, t (11, 19) (q23; p13.1), -12, + mar/46, XY. Next-generation sequencing showed a fusion of MLL exon 7 to exon 2 of ELL. A diagnosis of MLL-ELL-positive AML (M2 subtype) with subcutaneous MS was made. CONCLUSION: MLL-ELL-positive AML with MS is a rare clinical entity. Additional research is needed to elucidate the molecular mechanisms of the pathogenesis of MS.

Pharmacological inhibition of the mixed lineage leukemia 1-menin interaction aggravates acute kidney injury induced by folic acid and ischemia-reperfusion in mice.

Mixed lineage leukemia 1 (MLL1) is a methyltransferase that induces histone H3 lysine 4 trimethylation (H3K4me3) and partially exerts its untoward functional effects by interacting with multiple subunits including menin and WD repeat-containing protein 5 (WDR5). In this study, we investigated the role and mechanisms of MLL1 in murine models of acute kidney injury induced by folic acid (FA) and ischemia-reperfusion. Injury to the kidney elevated the expression of MLL1, menin, WDR5, and H3K4Me3, which was accompanied by increased serum creatinine and blood urea nitrogen, renal tubular injury, and apoptosis. Pharmacological inhibition of MLL1 activity with MI503 to disrupt the interaction between MLL1 with menin further increased serum creatinine and blood urea nitrogen levels, enhanced expression of neutrophil gelatinase-associated lipocalin and kidney injury molecule-1, and induced more apoptosis in the kidney following FA and ischemia-reperfusion injury. In contrast, MI503 treatment decreased the expression of vimentin and proliferating cell nuclear antigens. Similarly, treatment with MM102 to disrupt the interaction between MLL1 and WDR5 also worsened renal dysfunction, aggravated tubular cell injury, increased apoptosis, and inhibited cellular dedifferentiation and proliferation in mice following FA injection. Moreover, MI503 inhibited FA-induced phosphorylation of epidermal growth factor receptor, signal transducer and activator of transcription 3, and extracellular signal-regulated kinase-1/2 in injured kidneys. Collectively, these data suggest that MLL1 contributes to renal protection and functional recovery and promotes renal regeneration through a mechanism associated with activation of the epidermal growth factor receptor signaling pathway.NEW & NOTEWORTHY Mixed lineage leukemia 1 (MLL1) is a methyltransferase that induces histone H3 lysine 4 trimethylation and exerts its functional roles by interacting with multiple subunits. In this study, we demonstrated that inhibition of MLL1 activity by MI503 or MM102 aggravated renal injury and apoptosis and suppressed renal tubular cell dedifferentiation and proliferation, suggesting that MLL1 activation during acute kidney injury acts as an intrinsic protective mechanism to mediate renal tubular cell survival and regeneration.

AFF4 globally affects the release of paused RNA polymerase II in HEL cells.

P-TEFb, a heterodimer of the kinase CDK9 and Cyclin T1, is a critical regulator of promoter-proximal pause release of Pol II in metazoans. It is capable of forming three larger complexes, including the super elongation complex (SEC), the BRD4/P-TEFb complex and the 7SK snRNP. In the SEC or the BRD4/P-TEFb complex, P-TEFb is enzymatically active, while in the 7SK snRNP, its activity is inhibited. The SEC consists of AFF1 or 4, ENL or AF9, ELL1, 2 or 3 and EAF1 or 2 in addition to P-TEFb, the only subunit with catalytic activity, and the noncatalytic subunits have been found to be able to regulate pause release through P-TEFb. We and others recently found that AFF1, ENL and AF9 are capable of regulating transcriptional initiation, but it is unknown yet whether AFF4 is also capable of doing so. With respect to the gene regulation selectivity of the SEC and the BRD4/P-TEFb complex, one recent study showed that in human DLD-1 cells, the SEC only regulates pause release of heat shock (HS) genes, whereas the BRD4/P-TEFb complex regulates pause release of the rest of the genes. However, it is unclear whether those mechanisms are general. In this study for the purpose of further understanding the role of AFF4 in transcriptional regulation, we found that AFF4 knockdown by RNA interference in human HEL cells decreased not only cellular level but also global chromatin occupancy of CTD serine 2 phosphorylated Pol II. Direct target genes of AFF4 were identified by RNA-seq and CUT&Tag. Notably, we found by ChIP-seq and PRO-seq that AFF4 loss also increased promoter-proximal pause of Pol II on several hundred HS and thousands of non-HS genes. Mechanistically, AFF4 promotes pause release likely by facilitating the binding of P-TEFb to Pol II. These results suggest that extent of the impact of AFF4 on pause release is likely to be context-dependent or cell-type dependent.

Ruxolitinib inhibits the proliferation and induces the apoptosis of MLL-r ALL cells through inactivating JAK/STAT signaling pathway.

Background: The childhood patients with mixed-lineage leukemia rearrangement (MLL-r) gene have worse outcome than non-MLL, and thus often treated with high-risk chemotherapy regimens, so targeted therapy is important for this type of leukemia. This purpose of study was to explore the effects of ruxolitinib on the proliferation, apoptosis, and cell cycle of Nalm-6 cells. Methods: In this study, human acute lymphoblastic leukemia (ALL) cell line Nalm-6 was used as the research object. By constructing an MLL overexpression vector to transfect Nalm-6 cells, exogenous JAK2/STAT3 signal pathway inhibitor ruxolitinib was applied to observe the proliferation, apoptosis, and cell cycle changes of the transfected Nalm-6 cells. Western blot was performed to determine the proteins (MLL-BP, JAK, STAT) involved in the mechanism of action of MLL-r leukemia. CCK8 assay and flow cytometry (FCM) were used for testing the proliferation and apoptosis among MLL-BP transfected Nalm-6 cells. Results: Firstly, we determine the IC50 of ruxolitinib on Nalm-6 cells. Secondly, FCM and CCK8 showed that ruxolitinib dosedependentlyinhibits proliferation of Nalm-6 cells by blocking the cell cycle at G0/G1 phase. In addition, FCM showed that ruxolitinib promoted the apoptosis of MLL-BP transfected Nalm-6 cells. Mechanistically, ruxolitinib inactivated JAK/STAT signaling pathway in MLL-BP transfected Nalm-6 cells, mediating ruxolitinib's inhibition of cell proliferation, and inducing apoptosis. Finally, ruxolitinib significantly inhibited the proliferation of MLL-r ALL cells and promoted their apoptosis. Conclusions: These data provide compelling evidence that ruxolitinib is a promising agent against MLL-r leukemia cell line. However, it needs going through multiple more steps to confirm before it can be an option in clinical practice.

Taspase1 Facilitates Topoisomerase IIß-Mediated DNA Double-Strand Breaks Driving Estrogen-Induced Transcription.

The human protease Taspase1 plays a pivotal role in developmental processes and cancerous diseases by processing critical regulators, such as the leukemia proto-oncoprotein MLL. Despite almost two decades of intense research, Taspase1's biology is, however, still poorly understood, and so far its cellular function was not assigned to a superordinate biological pathway or a specific signaling cascade. Our data, gained by methods such as co-immunoprecipitation, LC-MS/MS and Topoisomerase II DNA cleavage assays, now functionally link Taspase1 and hormone-induced, Topoisomerase IIß-mediated transient DNA double-strand breaks, leading to active transcription. The specific interaction with Topoisomerase IIα enhances the formation of DNA double-strand breaks that are a key prerequisite for stimulus-driven gene transcription. Moreover, Taspase1 alters the H3K4 epigenetic signature upon estrogen-stimulation by cleaving the chromatin-modifying enzyme MLL. As estrogen-driven transcription and MLL-derived epigenetic labelling are reduced upon Taspase1 siRNA-mediated knockdown, we finally characterize Taspase1 as a multifunctional co-activator of estrogen-stimulated transcription.

MLL4 Regulates the Progression of Non-Small-Cell Lung Cancer by Regulating the PI3K/AKT/SOX2 Axis.

PURPOSE: Mixed-lineage leukemia protein 4 (MLL4/KMT2D) is a histone methyltransferase, and its mutation has been reported to be associated with a poor prognosis in many cancers, including lung cancer. We investigated the function of MLL4 in lung carcinogenesis. Materials and Methods: RNA sequencing (RNA-seq) in A549 cells transfected with control siRNA or MLL4 siRNA was performed. Also, we used EdU incorporation assay, colony formation assays, growth curve analysis, transwell invasion assays, immunohistochemical staining, and in vivo bioluminescence assay to investigate the function of MLL4 in lung carcinogenesis. RESULTS: We found that MLL4 expression was downregulated in non-small cell lung cancer (NSCLC) tissues compared to adjacent normal tissues and tended to decrease with disease stage progression. We analyzed the transcriptomes in control and MLL4- deficient cells using high-throughput RNA deep sequencing (RNA-seq) and identified a cohort of target genes, such as SOX2, ATF1, FOXP4, PIK3IP1, SIRT4, TENT5B, and LFNG, some of which are related to proliferation and metastasis. Our results showed that low expression of MLL4 promotes NSCLC cell proliferation and metastasis and is required for the maintenance of NSCLC stem cell properties. CONCLUSION: Our findings identify an important role of MLL4 in lung carcinogenesis through transcriptional regulation of PIK3IP1, affecting the PI3K/AKT/SOX2 axis, and suggest that MLL4 could be a potential prognostic indicator and target for NSCLC therapy.

An Alternative Splicing Variant of the Mixed-Lineage Leukemia 5 Protein Is a Cellular Adhesion Receptor for ScaA of Orientia tsutsugamushi.

Scrub typhus is a mite-borne disease caused by the obligately intracellular bacterium Orientia tsutsugamushi. We previously demonstrated that ScaA, an autotransporter membrane protein of O. tsutsugamushi, is commonly shared in various genotypes and involved in adherence to host cells. Here, we identified a mixed-lineage leukemia 5 (MLL5) mammalian trithorax group protein as a host receptor that interacts with ScaA. MLL5, identified by yeast two-hybrid screening, is an alternative splicing variant of MLL5 (vMLL5) which contains 13 exons with additional intron sequences encoding a tentative transmembrane domain. Indeed, vMLL5 is expressed on the plasma membrane as well as in intracellular compartments in eukaryotic cells and colocalized with adherent O. tsutsugamushi. In addition, ScaA-expressing Escherichia coli showed significantly increased adherence to vMLL5-overexpressing cells compared with vector control cells. We mapped the C-terminal region of the passenger domain of ScaA as a ligand for vMLL5 and determined that the Su(var)3-9, Enhancer of zeste, Trithorax (SET) domain of MLL5 is an essential and sufficient motif for ScaA binding. We observed significant and specific inhibition of bacterial adhesion to host cells in competitive inhibition assays using the C-terminal fragment of ScaA or the SET domain of vMLL5. Moreover, immunization with the C-terminal fragment of ScaA provided neutralizing activity and protective immunity against lethal challenge with O. tsutsugamushi as efficiently as vaccination with the whole passenger domain of ScaA. These results indicate that vMLL5 is a novel cellular receptor for ScaA-mediated adhesion of O. tsutsugamushi and facilitates bacterial adhesion to host cells, thereby enhancing bacterial infection. IMPORTANCE O. tsutsugamushi is a mite-borne pathogen that causes scrub typhus. As an obligately intracellular pathogen, its adhesion to and invasion of host cells are critical steps for bacterial growth. However, the molecular basis of the bacterial ligand and host receptor interaction is poorly defined. Here, we identified a splicing variant of MLL5 (vMLL5) as a cellular adhesion receptor of ScaA, an outer membrane autotransporter protein of O. tsutsugamushi. We mapped the interacting domains in the bacterial ligand and host receptor and confirmed their functional interaction. In addition, immunization with the C-terminal region of ScaA, which involves an interaction with the SET domain of vMLL5, not only induces enhanced neutralizing antibodies but also provides protective immunity against lethal challenge with O. tsutsugamushi.

Inhibition of MLL1-menin interaction attenuates renal fibrosis in obstructive nephropathy.

Mixed lineage leukemia 1 (MLL1), a histone H3 lysine 4 (H3K4) methyltransferase, exerts its enzymatic activity by interacting with menin and other proteins. It is unclear whether inhibition of the MLL1-menin interaction influences epithelial-mesenchymal transition (EMT), renal fibroblast activation, and renal fibrosis. In this study, we investigated the effect of disrupting MLL1-menin interaction on those events and mechanisms involved in a murine model of renal fibrosis induced by unilateral ureteral obstruction (UUO), in cultured mouse proximal tubular cells and renal interstitial fibroblasts. Injury to the kidney increased the expression of MLL1 and menin and H3K4 monomethylation (H3K4me1); MLL1 and menin were expressed in renal epithelial cells and renal interstitial fibroblasts. Inhibition of the MLL1-menin interaction by MI-503 administration or siRNA-mediated silencing of MLL1 attenuated UUO-induced renal fibrosis, and reduced expression of α-smooth muscle actin (α-SMA) and fibronectin. These treatments also inhibited UUO-induced expression of transcription factors Snail and Twist and transforming growth factor ß1 (TGF-ß1) while expression of E-cadherin was preserved. Moreover, treatment with MI-503 and transfection with either MLL siRNA or menin siRNA inhibited TGF-ß1-induced upregulation of α-SMA, fibronectin and Snail, phosphorylation of Smad3 and AKT, and downregulation of E-cadherin in cultured renal epithelial cells. Finally, MI-503 was effective in abrogating serum or TGFß1-induced transformation of renal interstitial fibroblasts to myofibroblasts in vitro. Taken together, these results suggest that targeting disruption of the MLL1-menin interaction attenuates renal fibrosis through inhibition of partial EMT and renal fibroblast activation.

MLL1:EZH2 Ratio in Uterine Secretions and Endometrial Receptivity in Patients with Endometriosis.

OBJECTIVE: To establish a novel approach for diagnosing endometriosis (EM) in patients with impaired endometrial receptivity. METHOD: Mixed lineage leukemia 1 (MLL1) and enhancer of zeste homolog 2 (EZH2) levels were analyzed. The MLL1:EZH2 ratio in identifying impaired endometrial receptivity has been established and validated. RESULTS: In normal endometrial tissue, the MLL1:EZH2 ratio increased significantly in the midsecretory phase, compared with that in the proliferative phase. In the midsecretory phase, the MLL1:EZH2 ratio in endometrial tissues and uterine secretions accurately identifies patients with EM who have impaired endometrial receptivity. In the validation group, the sensitivity and specificity of the MLL1:EZH2 ratio in the uterine secretions of the midsecretory phase, in diagnosing patients EM who have impaired endometrial receptivity, were 100% and 96.55%, respectively. CONCLUSIONS: The MLL1:EZH2 ratio in uterine secretions of the midsecretory phase may serve as a marker to diagnose EM in patients with impaired endometrial receptivity.

The Histone Deacetylase Inhibitor I1 Induces Differentiation of Acute Leukemia Cells With MLL Gene Rearrangements via Epigenetic Modification.

Acute leukemia (AL) is characterized by excessive proliferation and impaired differentiation of leukemic cells. AL includes acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Previous studies have demonstrated that about 10% of AML and 22% of ALL are mixed lineage leukemia gene rearrangements (MLLr) leukemia. The prognosis of MLLr leukemia is poor and new therapeutics are urgently needed. Differentiation therapy with all-trans-retinoic acid (ATRA) has prolonged the 5-years disease-free survival rate in acute promyelocytic leukemia (APL), a subtype of AML. However, the differentiation therapy has not been effective in other acute leukemia. Here, we aim to explore the cell differentiation effect of the potent HDACs inhibitor, I1, and the possible mechanism on the MLLr-AML and MLLr-ALL cells (MOLM-13, THP-1, MV4-11 and SEM). It is shown that I1 can significantly inhibit the proliferation and the colony-forming ability of MOLM-13, THP-1, MV4-11 and SEM cells by promoting cell differentiation coupled with cell cycle block at G0/G1 phase. We show that the anti-proliferative effect of I1 attributed to cell differentiation is most likely associated with the HDAC inhibition activity, as assessed by the acetylation of histone H3 and H4, which may dictates the activation of hematopoietic cell lineage pathway in both MOLM-13 and THP-1 cell lines. Moreover, the activity of HDAC inhibition of I1 is stronger than that of SAHA in MOLM-13 and THP-1 cells. Our findings suggest that I1, as a chromatin-remodeling agent, could be a potent epigenetic drug to overcome differentiation block in MLLr-AL patients and would be promising for the treatment of AL.

JMJD1C-regulated lipid synthesis contributes to the maintenance of MLL-rearranged acute myeloid leukemia.

Mixed Lineage Leukemia rearranged acute myeloid leukemia (MLLr AML) predicts a poor prognosis. Histone demethylase JMJD1C is a potential druggable target of MLLr AML. However, little is known about how JMJD1C contributes to MLLr AML. Here we found that JMJD1C regulates lipid synthesis-associated genes including FADS2, SCD in MLLr AML cells. Lipid synthesis-associated protein FABP5 was identified as a specific interacting protein of JMJD1C and binds to the jumonji domain of JMJD1C. FABP5 also regulates JMJD1C mRNA and protein expression. JDI-10, a small molecular inhibitor of JMJD1C identified by us, represses MLLr AML cells, induces apoptosis, and decreases JMJD1C-regulated lipid synthesis genes. Moreover, JDI-10 mediated suppression of MLLr AML cells can be rescued by palmitic acid, oleic acid, or recombinant FABP5. In summary, we identified that JMJD1C-regulated lipid synthesis contributes to the maintenance of MLLr AML. Lipid synthesis repression may represent a new direction for the treatment of MLLr AML.

DOT1L activity in leukemia cells requires interaction with ubiquitylated H2B that promotes productive nucleosome binding.

DOT1L methylates histone H3 lysine 79 during transcriptional elongation and is stimulated by ubiquitylation of histone H2B lysine 120 (H2BK120ub) in a classical trans-histone crosstalk pathway. Aberrant genomic localization of DOT1L is implicated in mixed lineage leukemia (MLL)-rearranged leukemias, an aggressive subset of leukemias that lacks effective targeted treatments. Despite recent atomic structures of DOT1L in complex with H2BK120ub nucleosomes, fundamental questions remain as to how DOT1L-ubiquitin and DOT1L-nucleosome acidic patch interactions observed in these structures contribute to nucleosome binding and methylation by DOT1L. Here, we combine bulk and single-molecule biophysical measurements with cancer cell biology to show that ubiquitin and cofactor binding drive conformational changes to stimulate DOT1L activity. Using structure-guided mutations, we demonstrate that ubiquitin and nucleosome acidic patch binding by DOT1L are required for cell proliferation in the MV4; 11 leukemia model, providing proof of principle for MLL targeted therapeutic strategies.

Adult Acute Myeloid Leukemia with the KMT2A-Mixed Lineage Leukemia T10 Fusion: An Analysis of 10 Cases Showed Common Features and Frequent Mutations in the RAS Signaling Pathway.

Mixed lineage leukemia (MLL) T10 is a relatively rare partner for the KMT2A lysine (K)-specific methyltransferase 2A gene. The common features and coexisting mutations of acute myeloid leukemia (AML) patients with KMT2A-MLLT10 remain unknown. In this study, 10 adult AML patients with KMT2A-MLLT10 fusions were picked up from 496 AML patients by using RT-polymerase chain reaction (PCR) and/or fluorescence in situ hybridization, and then screened for mutations in the 49 genes panel with next-generation sequencing and PCR, followed by direct Sanger sequencing. Of the 10 unique individuals identified, 6 were male and 4 were female (M:F ratio, 1.5:1) with ages ranging from 19 to 52 years (median 39.5 years). Most (90%, 9/10) patients with KMT2A-MLLT10 were accompanied by additional mutations. Twelve mutated genes were detected, averaging 2.1 mutations per patient (range, 0-4). The most frequently mutated gene was NRAS (n = 5). Clinical and laboratory data pointed to common features: French American British-M5 subtype (n = 7), a high rate of relapse, and biomarkers CD33 (n = 10), CD117 (n = 9), CD13 (n = 8), and CD64 (n = 8). Overall, most patients harbored at least one mutation. A high incidence of mutations affecting the RAS signaling pathway or RAS regulating components was found in 50% (5/10) patients. The overall survival is about 12.0 months. Allogeneic-hematopoietic stem cell transplantation trends to improve survival in selected patients.

Research advances of small molecule inhibitors in the treatment of mixed lineage leukemia / 药学学报

Acute leukemia (AL) is a kind of malignant clonal disease of hematopoietic stem cells. Rearrangement of mixed lineage leukemia (MLL) gene can be observed in about 5%-10% of AL patients. Currently, AL patients with MLL-rearrangements (MLL-r) lack effective treatment and are usually associated with poor prognoses. Recent studies have shown that many epigenetic regulators are directly or indirectly involved in the occurrence and development of AL carrying MLL-r (MLL), which provides a biological basis for the use of epigenetic regulation strategies to treat MLL. In this review, we start from the epigenetic regulation mechanism of MLL, and select representative drug targets to briefly analyze the relationship between each target and MLL and summarize the development progress of their inhibitors, hoping to provide reference for the subsequent research and development of drugs for the treatment of MLL.

GRWD1-WDR5-MLL2 Epigenetic Complex Mediates H3K4me3 Mark and Is Essential for Kaposi's Sarcoma-Associated Herpesvirus-Induced Cellular Transformation.

Infection by Kaposi's sarcoma-associated herpesvirus (KSHV) is causally associated with numerous cancers. The mechanism of KSHV-induced oncogenesis remains unclear. By performing a CRISPR-Cas9 screening in a model of KSHV-induced cellular transformation of primary cells, we identified epigenetic regulators that were essential for KSHV-induced cellular transformation. Examination of TCGA data sets of the top 9 genes, including glutamate-rich WD repeat containing 1 (GRWD1), a WD40 family protein upregulated by KSHV, that had positive effects on cell proliferation and survival of KSHV-transformed cells (KMM) but not the matched primary cells (MM), uncovered the predictive values of their expressions for patient survival in numerous types of cancer. We revealed global epigenetic remodeling including H3K4me3 epigenetic active mark in KMM cells compared to MM cells. Knockdown of GRWD1 inhibited cell proliferation, cellular transformation, and tumor formation and caused downregulation of global H3K4me3 mark in KMM cells. GRWD1 interacted with WD repeat domain 5 (WDR5), the core protein of H3K4 methyltransferase complex, and several H3K4me3 methyltransferases, including myeloid leukemia 2 (MLL2). Knockdown of WDR5 and MLL2 phenocopied GRWD1 knockdown, caused global reduction of H3K4me3 mark, and altered the expression of similar sets of genes. Transcriptome sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) analyses further identified common and distinct cellular genes and pathways that were regulated by GRWD1, WDR5, and MLL2. These results indicate that KSHV hijacks the GRWD1-WDR5-MLL2 epigenetic complex to regulate H3K4me3 methylation of specific genes, which is essential for KSHV-induced cellular transformation. Our work has identified an epigenetic complex as a novel therapeutic target for KSHV-induced cancers. IMPORTANCE By performing a genome-wide CRISPR-Cas9 screening, we have identified cellular epigenetic regulators that are essential for KSHV-induced cellular transformation. Among them, GRWD1 regulates epigenetic active mark H3K4me3 by interacting with WDR5 and MLL2 and recruiting them to chromatin loci of specific genes in KSHV-transformed cells. Hence, KSHV hijacks the GRWD1-WDR5-MLL2 complex to remodel cellular epigenome and induce cellular transformation. Since the dysregulation of GRWD1 is associated with poor prognosis in several types of cancer, GRWD1 might also be a critical driver in other viral or nonviral cancers.

ZNF521 Enhances MLL-AF9-Dependent Hematopoietic Stem Cell Transformation in Acute Myeloid Leukemias by Altering the Gene Expression Landscape.

Leukemias derived from the MLL-AF9 rearrangement rely on dysfunctional transcriptional networks. ZNF521, a transcription co-factor implicated in the control of hematopoiesis, has been proposed to sustain leukemic transformation in collaboration with other oncogenes. Here, we demonstrate that ZNF521 mRNA levels correlate with specific genetic aberrations: in particular, the highest expression is observed in AMLs bearing MLL rearrangements, while the lowest is detected in AMLs with FLT3-ITD, NPM1, or CEBPα double mutations. In cord blood-derived CD34+ cells, enforced expression of ZNF521 provides a significant proliferative advantage and enhances MLL-AF9 effects on the induction of proliferation and the expansion of leukemic progenitor cells. Transcriptome analysis of primary CD34+ cultures displayed subsets of genes up-regulated by MLL-AF9 or ZNF521 single transgene overexpression as well as in MLL-AF9/ZNF521 combinations, at either the early or late time points of an in vitro leukemogenesis model. The silencing of ZNF521 in the MLL-AF9 + THP-1 cell line coherently results in an impairment of growth and clonogenicity, recapitulating the effects observed in primary cells. Taken together, these results underscore a role for ZNF521 in sustaining the self-renewal of the immature AML compartment, most likely through the perturbation of the gene expression landscape, which ultimately favors the expansion of MLL-AF9-transformed leukemic clones.