Mixed Lineage Leukemia Research Articles

HMGB1 Interacts with the MLL-AF4 Fusion Complex to Regulate Pro-Leukemic Gene Transcription in Infant Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) in infants carries a poor prognosis and is characterized by cytogenetic rearrangements producing abnormal MLL fusion genes. Clinically effective targeting of the MLL fusion heterocomplex remains challenging, and therapeutic options remain limited. We have observed that the reduced isoform of HMGB1, a chromatin architectural protein that stabilizes DNA and facilitates transcription, is selectively over-expressed in the nuclei of infant MLL-ALL cells. In this study, we generated an HMGB1 siRNA knockdown in primary MLL-ALL cells from 3 infants to test our hypothesis that HMGB1-MLL interactions regulate pro-leukemic gene expression and represent a rational therapeutic target.CD19-selected leukemic blasts were isolated from the cryopreserved bone marrow or peripheral blood specimens of 3 infants with cytogenetically confirmed MLL-AF4 rearrangements. HMGB1 knockdown was confirmed by comparing HMGB1 mRNA and protein expression, by qPCR and Western Blot, in cells transfected with HMGB1 vs. control sequence siRNA. First, determined whether HMGB1 knockdown affected expression of the MLL fusion gene itself, by comparing MLL-AF4 mRNA and protein levels 72 hours after siRNA transfection. HMGB1 knockdown produced a 2.8 (± 0.55)- fold decrease in MLL-AF4 mRNA expression by qPCR (p<0.05), with a corresponding decrease in MLL-AF4 fusion protein expression by Western Blot, in each of the 3 specimens.Next, we determined whether HMGB1 binds functionally relevant regions of the MLL gene. We developed an electrophoretic mobility assay (EMSA) to compare the mobility of lysates from control vs. HMGB1 siRNA-treated infant MLL-ALL cells when mixed with biotinylated oligonucleotides spanning the transcriptionally active domains of MLL1. In each of 3 primary infant MLL-ALL cells, we detected a consistent gel-shift pattern on SDS-PAGE, in wild-type and control siRNA lysates, with oligonucleotides spanning exons 6-9- where many MLL-AF4 fusions occur. The gel-shift was completely abrogated in HMGB1 siRNA lysates. We then compared the expression of MLL target genes involved in leukemic transformation, by qPCR, in infant MLL-ALL cells treated with HMGB1 vs. control siRNA. We observed a significant (p<0.01) reduction in expression of MEIS1 (5.8 ± 2.2-fold decrease), HOXA7 (4.3 ± 0.4-fold decrease) and HOXA9 (3.7 ± 1.5-fold decrease) in infant MLL-ALL cells treated with HMGB1 vs. control siRNA. These data confirmed a role for HMGB1 in MLL gene/target gene regulation at the DNA level.Finally, we considered whether HMGB1, as a scaffold protein, could interact directly with the MLL fusion heterocomplex at the protein level. We immunoprecipitated HMGB1 from the nuclear fraction of wild-type primary infant MLL-ALL cells (n=3 patients), then probed the pull-down for N-terminal MLL (MLLn), C-terminal MLL (MLLc), the MLLn-AF4 fusion, the MLLn-ENL fusion, and the MLL-associated histone 3 methyltransferase DOT1L. MLLn and MLLn-AF4 were strongly detected in all HMGB1 immunoprecipitates. Individual and sequential co-immunoprecipitation of HMGB1 with MLL-AF4 and DOT1L in revealed loss of known complex formation between MLL-AF4 and DOT1L following HMGB1 knockdown. This was accompanied by a 3.4 (± 0.9)-fold decrease in DOT1L mRNA expression (p<0.001) by qPCR and a complete loss of histone 3k79me2 protein expression by Western blot.Taken together, these data suggest a central role for the fully reduced isoform of HMGB1, found in high abundance in infant ALL nuclei, in the formation of the MLL-AF4 transcription complex- including for the stable recruitment of DOT1L and H3K79me2, and in the regulation of MLL target genes such as HOXA9 and MEIS1. We are currently conducting chromatin immunoprecipitation and sequencing studies to identify methylation marks, particularly at H3K79me2, impacted by HMGB1 knockdown in infant ALL cells. We hope these studies will directly inform the development of small molecule inhibitors that specifically disrupt the binding sites and capacities of HMGB1 with MLL, which could synergize with the effects of methyltransferase inhibitors to more completely silence leukemic gene expression in infant ALL and improve the prognosis of this devastating disease. DisclosuresNo relevant conflicts of interest to declare.

Genome-Wide CRISPR-Cas9 Screen Identifies Sensitizers to LSD1 Inhibition in MLL-Translocated Human AML Cells

Lysine-specific demethylase (LSD1, also known as KDM1A) is an epigenetic regulator that has recently emerged as a potential therapeutic target in acute myeloid leukemia (AML). It is a flavin dependent monoamine oxidase which can demethylate monomethyl or dimethyl lysine 4 of histone H3. Pharmacological inhibition of LSD1 induces differentiation of blast cells in MLL-translocated AML and has shown significant promise in pre-clinical studies. With LSD1 inhibitors advancing through early-phase clinical trials, there is a strong pre-clinical rationale for the identification of genes whose protein products collaborate with LSD1 to retard differentiation in cancer and which could potentially be targeted in combination therapies for enhanced therapeutic benefit.To identify potential drug-gene synthetic lethal interactions, we performed a genome wide loss-of-function CRISPR-Cas9 screen in human THP1 AML cells treated with a potent and selective tranylcypromine-derivative LSD1 inhibitor trans-N-((2-methoxypyridin-3-yl)methyl)-2-phenylcyclopropan-1-amine (OG86). THP1 cells exhibit a t(9;11) MLL gene rearrangement and display similar phenotypic and functional responses to those observed in primary MLL-translocated AML cells following LSD1 inhibition. The cells were transduced with lentiviral human CRISPR knockout (hGeCKOv2) library containing 122,411 sgRNAs targeting 19,050 protein coding genes and 1864 miRNA precursors in the human genome. The transduced cells were divided into two groups, treated with either DMSO or 250nM OG86 and maintained in culture for ~10 population doublings. To investigate sgRNA representation in the cell population harvested at different stages of the screen, sgRNA cassettes were PCR amplified from genomic DNA and deep sequenced; negatively selected genes were identified with the MAGeCK computational algorithm. After successful initial quality assessment of the screen, we next searched for genes selectively depleted in OG86-treated versus DMSO-treated THP1 cells in samples collected on Day 15 and Day 18. At a false discovery rate of 7.5% there were 10 expressed genes whose sgRNA representation was depleted at both time points. In particular, these included two genes coding for components of the MTOR signaling pathway: MTOR associated protein, LST8 homolog (MLST8) and Ras-related GTP-binding protein A (RRAGA). In the Day 18 comparison, an additional MTOR pathway gene LAMTOR2 scored among the ten most depleted. MLST8 is a core component of TORC1/TORC2 complex and RRAGA is involved in the activation of TORC1 by amino acids. Recruitment of both RAG proteins and TORC1 to lysosomal membranes in response to amino acids, and the consequent activation of TORC1 signalling, requires the trimeric Ragulator complex, of which LAMTOR2 is a member.Based on our screen, we hypothesized that THP1 AML cells exposed to pharmacologic inhibition of LSD1 exhibit increased sensitivity to concomitant inhibition of the amino acid sensing component of the TORC1 pathway.Using genetic knockdown and pharmacological inhibition strategies, we then validated our screen hits in combination with LSD1 inhibition in targeting human AML cells. RNAi based knockdown of RRAGA, LAMTOR2 and MLST8 in combination with LSD1 inhibition were found to promote myeloid differentiation and reduce cell proliferation in THP1 cells. Interestingly the mTORC1 pathway inhibitor everolimus (RAD001) showed at least additive effect in combination with OG86 to decrease THP1 cell proliferation and promote immunophenotypic differentiation. Comparison of transcription changes in combined versus single treatment conditions by RNA-seq analysis further confirmed a more extensive and wide-ranging upregulation of a myeloid differentiation program upon concomitant inhibition of LSD1 and mTORC1 pathway. In vitro studies performed in primary patient AML cells gave similar results. Finally, in vivo studies using AML patient-derived xenograft mouse model confirmed that combination treatment promotes a strong myeloid differentiation program. In conclusion, we report here that inhibition of mTORC1 sensitizes human MLL-translocated AML cells to LSD1 inhibitor-mediated differentiation therefore highlighting a novel combination approach for evaluation in clinical trials. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of PRMT1 Mediated FLT3 Arginine Methylation As a Potent Therapeutic Strategy for MLL-r ALL

Mixed-lineage leukemia-rearranged (MLL-r) ALL, seen in 70% of infant ALL, has a dismal prognosis compared to those with wild type MLL1 gene. Transcriptional profiling has identified Fms-like receptor tyrosine kinase 3 (FLT3) as one of the most significantly upregulated genes in MLL-r ALL. The highly expressed FLT3 protein is activated by the autocrine ligand, making the kinase a therapeutic target. FLT3 tyrosine kinase inhibitors (TKIs) such as PKC412, although effective in kinase inhibition, partially impair survival of MLL-r ALL cells and clinical trial results are not promising, promoting us to ask whether FLT3 regulates the ALL cells survival also through a kinase-independent mechanism.Herein, we report the finding of dimethylated arginines on FLT3, detected through mass spectrometry analysis of a MLL-r ALL specimen and a MLL-r ALL line SEM. The most conserved and enriched of dimethylated arginines are residues R972/R973. Using home-made arginine methylation (R-Me) antibody, we found that PRMT1, which is responsible for most type I arginine methyltransferases activity, catalyzes FLT3 methylation. Immunoblot (IB) analysis validated the expression of FLT3 R-Me in MLL-r ALL samples (6 out of 6) and MLL-r ALL lines (4 out of 4). Analysis of the GEO dataset (GSE13204) revealed that PRMT1 mRNA levels are increased in MLL-r ALL relative to normal cells (MLL-r, n=70 vs. normal, n=73, p<0.0001). We studied FLT3 R-Me biological function using two approaches that specifically blocked FLT3 methylation levels: cells expressing FLT3 methylation deficient construct (R972/973K, arginine [R] to lysine [K]) exhibited reduced survival (BaF3: FLT3-WT 98.5±0.11% vs. R972/973K 71.5±0.53%, p=0.0004); knockdown of PRMT1 in SEM cells also had an inhibitory effect (siCtrl 95.1±0.1% vs. siPRMT1 74.7±0.5%, p=0.0007). Moreover, the type I arginine methyltransferase inhibitor MS023 (5 µM) treatment markedly induced apoptosis of primary ALL cells but spared normal counterparts from healthy donors (ALL: vehicle 10.4±0.4% vs. MS023 23.7±0.8%, n=4; p<0.0001; normal CD19+: 8.3±0.3% vs. 8.2±0.1%, n=3, p=0.86). Interestingly, inhibition of FLT3 methylation decreased FLT3 phosphorylation at tyrosine 969 (Y969) but not Y589/591 or Y842. Expression of R972/973K decreased FLT3 downstream signaling like phospho-STAT5 and -AKT to a greater extent than that of Y969F mutant (Y to phenylalanine [F] substitution, mimics loss of Y phosphorylation). Next, FLT3 WT, R972/973K or Y969F transduced primary MLL-r ALL cells were transplanted into NSGS mice for analysis of leukemia development (n=6/group). Mice transplanted with FLT3 Y969F MLL-r ALL had longer survival relative to FLT-WT injected animals (p=0.0031), and the median survival was further extended in mice injected with R972/973K mutant compared with FLT3 Y969F MLL-r ALL (p=0.0007). Additionally, PKC412 treatment alone did not alter FLT3 R-Me, and high FLT3 methylation level in SEM cells was not affected by FLT3 ligand stimulation, confirming that the function of R-Me is independent of FLT3 phosphorylation. Importantly, we observed that the combination of MS023 with PKC412 significantly induced a higher rate of apoptosis in primary MLL-r ALL cells compared with each drug alone (control, 10±0.43%, MS023, 21.1±1.2%, PKC412, 21.5±0.11%, combination, 39.8±2.9%, PKC412 vs combination, p<0.01, n=4). We further tested the effects of in vivo administration of MS023 plus PKC412 on primary MLL-r ALL cells xenografted in NSGS mice. Following engraftment >1% in peripheral blood, mice were subdivided into four groups and treated with vehicle, PKC412 (100 mg/kg, i.g.), MS023 (80 mg/kg, i.p, bid), or the combination (n=7/group) for 4 weeks. The BM tumor burden of CD45+ CD19+ cells was reduced in single drug-treated mice cohorts, with further reduction after combination treatment (vehicle, 94.4±0.5%, PKC412, 50.2±6.3%, MS023, 55.6±4.5%, combination, 30.7±4.9%, PKC412 vs. combination, p<0.001). Secondary transplantation of BM cells from mice receiving combination treatment resulted in significantly reduced BM engraftment at 16 weeks compared to PKC412 treatment alone (PKC412, 62.2±4.9%, combination, 8.4±5.1%, n=5, p<0.0001), indicating reduced leukemia initiating capacity.Our results support further exploring the molecular function of FLT3 R-Me. We will determine whether PRMT1 and FLT3 methylation are potential druggable targets in MLL-r ALL. DisclosuresKonopleva:Stemline Therapeutics: Research Funding.

Precision genomic and translational medicine for acute myeloid leukemia

Leukemia is a group of hematologic malignancy that has unfavorable prognosis and unclear mechanisms. In recent years, advances in leukemia research encompass the discovery of novel targets in acute myeloid leukemia drug resistance, epigenetic crosstalk in mixed lineage leukemia (MLL) leukemogenesis, genetic mechanisms of aggressive NK-cell leukemia, as well as the critical role of key epigenetic regulator in acute myeloid malignancy. Remarkably, researchers revealed that the histone modifying gene SETD2 as a new tumor suppressor and therapeutic target in patients with acute myeloid leukemia. Furthermore, low-dose chemotherapy as a frontline regiment in treating pediatric acute myeloid leukemia can substantially reduce the toxic side effects and treatment costs without impairing efficacy. Although advances in cancer genomics have greatly increased our understanding of the molecular characteristics in tumor biology, recent studies suggest that Darwinian evolution of intratumor heterogeneity represents a major challenge to develop therapeutic strategies to improve disease control. Researchers also dissected the distinct evolutionary dynamics under different chemotherapy regimens and the corresponding applications in the evaluation of treatment outcomes. Altogether, these efforts offered new opportunities for the development of acute myeloid leukemia diagnostics and therapeutics.

Therapeutic targeting potential of chromatin-associated proteins in MLL-rearranged acute leukemia.

Acute leukemias (AL) with a Mixed Lineage Leukemia (MLL) gene rearrangement (MLLr) represent a group of leukemic entities conferring intermediate to adverse prognoses. Multiple chromatin-associated proteins have been shown to play essential roles during the genesis of MLLr AL. Some chromatin-associated proteins function as negative regulators of MLLr AL whereas others are required for leukemic initiation or maintenance - the latter group constituting potential therapeutic targets. Most of the identified proteins have been functionally analyzed using experimental models with human/murine normal cells transformed by MLL-AF9 or other MLL fusion products, which may recapitulate most but not all aspects of human AML, such as immune system interactions - features of which the importance is rapidly emerging. Here, we review chromatin-associated proteins fundamental to MLLr AL development, highlighting those with targeting potential by small molecule inhibitors. In particular, we focus on synthetic targeting of multiple chromatin-associated proteins, a strategy that shows superior therapeutic efficacy and offers hope for overcoming drug resistance.

Isolation of MLL1 Inhibitory RNA Aptamers.

Mixed lineage leukemia proteins (MLL) are the key histone lysine methyltransferases that regulate expression of diverse genes. Aberrant activation of MLL promotes leukemia as well as solid tumors in humans, highlighting the urgent need for the development of an MLL inhibitor. We screened and isolated MLL1-binding ssRNAs using SELEX (Systemic Evolution of Ligands by Exponential enrichment) technology. When sequences in sub-libraries were obtained using next-generation sequencing (NGS), the most enriched aptamers—APT1 and APT2—represented about 30% and 26% of sub-library populations, respectively. Motif analysis of the top 50 sequences provided a highly conserved sequence: 5′-A[A/C][C/G][G/U][U/A]ACAGAGGG[U/A]GG[A/C] GAGUGGGU-3′. APT1, APT2, and APT5 embracing this motif generated secondary structures with similar topological characteristics. We found that APT1 and APT2 have a good binding activity and the analysis using mutated aptamer variants showed that the site information in the central region was critical for binding. In vitro enzyme activity assay showed that APT1 and APT2 had MLL1 inhibitory activity. Three-dimensional structure prediction of APT1-MLL1 complex indicates multiple weak interactions formed between MLL1 SET domain and APT1. Our study confirmed that NGS-assisted SELEX is an efficient tool for aptamer screening and that aptamers could be useful in diagnosis and treatment of MLL1-mediated diseases.

BET inhibitors reduce cell size and induce reversible cell cycle arrest in AML.

Inhibitors of the bromodomain and extraterminal domain family (BETi) offer a new approach to treat hematological malignancies, with leukemias containing mixed lineage leukemia rearrangements being especially sensitive due to a reliance on the regulation of transcription elongation. We explored the mechanism of action of BETi in cells expressing the t(8;21), and show that these compounds reduced the size of acute myeloid leukemia cells, triggered a rapid but reversible G0 /G1 arrest, and with time, cause cell death. Meta-analysis of PRO-seq data identified ribosomal genes, which are regulated by MYC, were downregulated within 3 hours of addition of the BETi. This reduction of MYC regulated metabolic genes coincided with the loss of mitochondrial respiration and large reductions in the glycolytic rate. In addition, gene expression analysis showed that transcription of BCL2 was rapidly affected by BETi but this did not cause dramatic increases in cell death. Cell cycle arrest, lowered metabolic activity, and reduced BCL2 levels suggested that a second compound was needed to push these cells over the apoptotic threshold. Indeed, low doses of the BCL2 inhibitor, venetoclax, in combination with the BETi was a potent combination in t(8;21) containing cells. Thus, BET inhibitors that affect MYC and BCL2 expression should be considered for combination therapy with venetoclax.

Disordered Regions of Mixed Lineage Leukemia 4 (MLL4) Protein Are Capable of RNA Binding.

Long non-coding RNAs (lncRNAs) are emerging as important regulators of cellular processes and are extensively involved in the development of different cancers; including leukemias. As one of the accepted methods of lncRNA function is affecting chromatin structure; lncRNA binding has been shown for different chromatin modifiers. Histone lysine methyltransferases (HKMTs) are also subject of lncRNA regulation as demonstrated for example in the case of Polycomb Repressive Complex 2 (PRC2). Mixed Lineage Leukemia (MLL) proteins that catalyze the methylation of H3K4 have been implicated in several different cancers; yet many details of their regulation and targeting remain elusive. In this work we explored the RNA binding capability of two; so far uncharacterized regions of MLL4; with the aim of shedding light to the existence of possible regulatory lncRNA interactions of the protein. We demonstrated that both regions; one that contains a predicted RNA binding sequence and one that does not; are capable of binding to different RNA constructs in vitro. To our knowledge, these findings are the first to indicate that an MLL protein itself is capable of lncRNA binding.

Chemosensitivity is differentially regulated by the SDF-1/CXCR4 and SDF-1/CXCR7 axes in acute lymphoblastic leukemia with MLL gene rearrangements

Although recent advances in chemotherapy have markedly improved outcome of acute lymphoblastic leukemia (ALL), infantile ALL with MLL gene rearrangements (MLL+ALL) is refractory to chemotherapy. We have shown that specific cytokines FLT3 ligand and TGFβ1 both of which are produced from bone marrow stromal cells synergistically induced MLL+ALL cells into chemo-resistant quiescence, and that treatment of MLL+ALL cells with inhibitors against FLT3 and/or TGFβ1 receptor partially but significantly converts them toward chemo-sensitive. In the present study, we showed that MLL+ALL cells expressed CXCR4 and CXCR7, both receptors for the same chemokine stromal cell derived factor-1 (SDF-1), but their biological events were differentially regulated by the SDF-1/CXCR4 and SDF-1/CXCR7 axes and particularly exerted an opposite effect for determining chemo-sensitivity of MLL+ALL cells; enhancement via the SDF-1/CXCR4 axis vs. suppression via the SDF-1/CXCR7 axis. Because cytosine-arabinoside-induced apoptosis of MLL+ALL cells was inhibited by pretreatment with the CXCR4 inhibitor but rather accelerated by pretreatment with the CXCR7 inhibitor, an application of the CXCR7 inhibitor may become a good treatment option in future for MLL+ALL patients. MLL+ALL has a unique gene profile distinguishable from other types of ALL and AML, and should be investigated separately in responses to biological active agents including chemokine inhibitors.

Bioflavonoids promote stable translocations between MLL-AF9 breakpoint cluster regions independent of normal chromosomal context: Model system to screen environmental risks.

Infant acute leukemias are aggressive and characterized by rapid onset after birth. The majority harbor translocations involving the MLL gene with AF9 as one of its most common fusion partners. MLL and AF9 loci contain breakpoint cluster regions (bcrs) with sequences hypothesized to be targets of topoisomerase II inhibitors that promote translocation formation. Overlap of MLL bcr sequences associated with both infant acute leukemia and therapy-related leukemia following exposure to the topoisomerase II inhibitor etoposide led to the hypothesis that exposure during pregnancy to biochemically similar compounds may promote infant acute leukemia. We established a reporter system to systematically quantitate and stratify the potential for such compounds to promote chromosomal translocations between the MLL and AF9 bcrs analogous to those in infant leukemia. We show bioflavonoids genistein and quercetin most biochemically similar to etoposide have a strong association with MLL-AF9 bcr translocations, while kaempferol, fisetin, flavone, and myricetin have a weak but consistent association, and other compounds have a minimal association in both embryonic stem (ES) and hematopoietic stem cell (HSC) populations. The frequency of translocations induced by bioflavonoids at later stages of myelopoiesis is significantly reduced by more than one log. The MLL and AF9 bcrs are sensitive to these agents and recombinogenic independent of their native context suggesting bcr sequences themselves are drivers of illegitimate DNA repair reactions and translocations, not generation of functional oncogenic fusions. This system provides for rapid systematic screening of relative risk, dose dependence, and combinatorial impact of multitudes of dietary and environmental exposures on MLL-AF9 translocations. Environ. Mol. Mutagen. 60: 154-167, 2019. © 2018 Wiley Periodicals, Inc.

The long noncoding RNA GAS8-AS1 suppresses hepatocarcinogenesis by epigenetically activating the tumor suppressor GAS8

Long noncoding RNAs (lncRNAs) are vital players in cancers, including hepatocellular carcinoma (HCC). We previously identified an lncRNA, GAS8-AS1, that is located in intron 2 of GAS8 However, its involvement in HCC is still largely unknown. In this study, we report that both GAS8-AS1 and its host gene GAS8 act as HCC tumor suppressors. We found that expression of GAS8-AS1 or GAS8 is significantly decreased in HCC tissues and is associated with a poor prognosis among HCC patients. Interestingly, lncRNA GAS8-AS1 could promote GAS8 transcription. We detected a CpG island in the GAS8 promoter, but lncRNA GAS8-AS1 did not affect DNA methylation at this GAS8 promoter site. Moreover, we identified two GAS8-AS1-interacting proteins, mixed-lineage leukemia 1 (MLL1), a histone 3 Lys-4 (H3K4) methyltransferase, and its partner WD-40 repeat protein 5 (WDR5). RNA pulldown, ChIP, and RNA immunoprecipitation assays revealed that GAS8-AS1 is required for maintaining the GAS8 promoter in an open chromatin state by recruiting the MLL1/WDR5 complex and for enhancing RNA polymerase II activity and GAS8 transcription. Of note, GAS8-AS1-dependent GAS8 hyperactivation inhibited malignant transformation of hepatocytes. Our results provide important insights into how lncRNA GAS8-AS1 suppresses HCC development and suggest potential strategies for treating patients with liver cancer.

MLL3 enhances the transcription of PD-L1 and regulates anti-tumor immunity

Tumor cells utilize the overexpression of the programmed death-1 ligand 1(PD-L1) to escape T-cell controlled immune-surveillance. The clinical therapy that dilapidates PD1 or PD-L1-mediated cancer tolerance has pushed out the need to uncover the molecular regulation of PD-L1 overexpression in the tumor cell. In this study, we identify histone methyltransferase mixed-lineage leukemia protein 3 (MLL3) as a critical regulator of PD-L1 in prostate cancer cells. MLL3 and PD-L1 were highly expressed in the metastatic cancer tissues, compared to the primary cancer tissues. Furthermore, their expressions were highly correlated in the cancer tissues in the databases of TCGA and Xiangya Hospital. We found that MLL3 bound to the enhancer of PD-L1. Depletion of MLL3 decreased the binding level of H3K4me1 in the enhancer of PD-L1 and Pol II Ser-5p in the promoter of PD-L1. Importantly, MLL3 depletion impaired the mouse xenograft growth and decreased the response to PD-L1 antibody treatment in mice. The findings extend the understanding of the biology regulation of PD-L1 transcription and identify the histone writer MLL3 in an important immune checkpoint, and give prominence to a hidden therapeutic target to conquer immune evasion by tumor cells.

A new complex rearrangement in infant ALL: t(X;11;17)(p11.2;q23;q12)

We present a case of an infant who developed pro-B acute lymphoblastic leukemia with a rare and complex MLL-translocation. Cytogenetic analysis of bone marrow cells at diagnosis showed a 46,XY,t(X;11)(p11.2;q23)[13]/46,XY[7] karyotype. Fluorescence in situ hybridization analysis using a break apart specific probes showed a split in the MLL gene. Long distance inverse-PCR and next generation sequencing analysis depicted a complex rearrangement t(X;11;17)(p11.2;q23;q12) involving MLL, MLLT6 and the genomic region Xp11.23, 41 bases upstream of the WDR45 gene. WDR45 encodes a beta-propeller protein essential for autophagocytosis. MLL rearrangements with involvement of Xp have not been previously described.

Distinct pathways affected by menin versus MLL1/MLL2 in MLL-rearranged acute myeloid leukemia

Disrupting the protein-protein interaction for molecularly targeted cancer therapeutics can be a challenging but promising strategy. Compounds that disrupt the interaction between menin, a chromatin-binding protein, and oncogenic mixed lineage leukemia fusion proteins (MLL-FPs) have shown significant promise in preclinical models of leukemia and have a high degree of selectivity for leukemia versus normal hematopoietic cells. Biochemical and structural studies demonstrate that, in addition to disrupting the menin-MLL-FP interaction, such compounds also inhibit menin-MLL1, menin-MLL2, and other menin-interacting proteins. Here, we address the degree to which disruption of menin-MLL-FP interactions or menin-MLL1/MLL2 interactions contribute to the antileukemia effect of menin inhibition. We show that Men1 deletion in MLL-AF9-transformed leukemia cells produces distinct cellular and molecular consequences compared with Mll1;Mll2 co-deletion and that compounds disrupting menin-MLL N-terminal interactions largely phenocopy menin loss. Moreover, we show that Mll1;Mll2-deficient leukemia cells exhibit enhanced sensitivity to menin interaction inhibitors, which is consistent with each regulating complementary genetic pathways. These data illustrate the heightened dependency of MLL-FPs on menin compared with wild-type MLL1/MLL2 for regulation of downstream target genes and argue that the predominant action of menin inhibitory compounds is through direct inhibition of MLL-FPs without significant contribution from MLL1/MLL2 inhibition.

Fluorescence in situ hybridization comparison of the prognostic factors in adult and pediatric acute lymphoblastic leukemia: A retrospective analysis of 282 cases.

Acute lymphoblastic leukemia (ALL) affects both children and adults. However, the prognosis of the two cohorts is quite different. The present aim was to review and evaluate one potential cause of why survival is poorer in adult ALL than pediatric ALL via fluorescence in situ hybridization (FISH). Clinical significant features were analyzed in 282 ALL cases. FISH was performed to study mixed lineage leukemia (MLL) translocation and the Philadelphia (Ph) chromosome in newly diagnosed patients, and was used to detect trisomy 4 or 10 and the translocation ETS leukemia-acute myeloid leukemia 1 (TEL-AML1) fusion gene. The overall survival/event-free survival (OS/EFS) outcome of adult ALL and pediatric ALL was analyzed using Kaplan-Meier analysis. Adult ALL had a higher median leukocyte count and lower hemoglobin level than pediatric ALL. FISH revealed that Ph positivity (Ph+) was associated with the high-risk feature of older age. In pediatric ALL, trisomy 4 or 10 was present in 71/207 cases (34.3%), while the TEL-AML1 fusion gene was present in 16/207 cases (7.7%). By contrast, there were very few such positive cases in adult ALL. Survival analysis revealed that, in adult ALL, the 3-year OS and EFS rates were higher in the Ph-negative group than in the Ph+ group. Adult or pediatric ALL is an independent prognostic factor of OS. The present analysis of the clinical and biological features between adult and pediatric ALL indicates that adult ALL has a poorer prognosis than pediatric ALL based on Ph+ status and presence of trisomy 4 or 10. Ph+ ALL is an independent prognosis factor of ALL. FISH may serve an important role in the comparison of prognostic factors in adult and pediatric ALL.

Abstract B21: Epigenetic modifiers MLL1 and JMJD3 regulate neuroblastoma tumorigenicity by maintaining a cancer stem cell-like population

Abstract High-risk neuroblastoma (NB) represents a major clinical challenge in pediatric oncology. Despite significant dose escalation of intense therapies, long-term survival for NB patients remains poor (&amp;lt;45%), and the disease accounts for almost 15% of all pediatric cancer deaths. Relapse of metastatic, drug-resistant disease and treatment-related toxicities mandates the development of new therapeutic strategies. Recently, we discovered a highly tumorigenic, chemoresistant, and self-renewing subpopulation in NB with features similar to cancer stem cells (CSCs). This G-CSF receptor (CD114)-expressing subpopulation can escape initial therapy and cause aggressively invasive relapsed disease. Therefore, developing direct targeting strategies and understanding for the molecular mechanisms maintaining this NB CSC-like subpopulation is essential to delineate effective therapeutics for NB patients. By using siRNA screening approach and low-density pathway arrays, we found that the epigenetic regulators mixed-lineage leukemia-1 (MLL1; KMT2A; a H3K4me3 methyltransferase) and Jumonji D3 (JMJD3; KDM6B; a H3K27me3 demethylase) are important epigenetic regulators for NB proliferation and growth. Interestingly, we found that epigenetic regulators MLL1 and JMJD3 are aberrantly expressed in CD114+ subpopulation and regulate the expression of the G-CSF receptor gene (CSF3R) itself, by maintaining active histone modifications at the promoter locus. Inhibition of MLL1 and JMJD3 with specific small-molecule inhibitors reverses the histone patterns at CSF3R promoter and blocks gene expression, induces apoptosis selectively in CD114+ cells, and inhibits overall NB proliferation in vitro. Furthermore, inhibiting MLL1 and JMJD3 leads to dramatic tumor regression (p&amp;lt;0.001) and reduction in incidences of metastasis (p&amp;lt;0.001) in vivo. Most interestingly, both of these inhibitors increase the overall survival of the treated mice in vivo. As expected, reduction in tumor size was significantly correlated with the reduction in tumor CD114+ cells. Taken together, these data highlight that: a) neuroblastoma is maintained by the complex interplay of epigenetic modifiers, and b) direct targeting of these epigenetic modifiers and combining this approach with current therapy is a novel therapeutic approach to high-risk NB. Citation Format: Saurabh Agarwal, Julie Tomolonis, Sanjeev Vasudevan, Jason Shohet. Epigenetic modifiers MLL1 and JMJD3 regulate neuroblastoma tumorigenicity by maintaining a cancer stem cell-like population [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B21.

CDKI-73: an orally bioavailable and highly efficacious CDK9 inhibitor against acute myeloid leukemia.

Acute myeloid leukemia (AML) is the most common form of acute leukemia with dismal long-term prognosis with age. The most aggressive subtype of AML is MLL-AML that is characterized by translocations of the mixed-lineage leukemia gene (MLL) and resistance to conventional chemotherapy. Cyclin dependent kinase 9 (CDK9) plays a crucial role in the MLL-driven oncogenic transcription, and hence, inhibiting activity of CDK9 has been proposed as a promising strategy for treatment of AML. We investigated the therapeutic potential of CDKI-73, one of the most potent CDK9 inhibitors, against a panel of AML cell lines and samples derived from 97 patients. CDKI-73 induced cancer cells undergoing apoptosis through transcriptional downregulation of anti-apoptotic proteins Bcl-2, Mcl-1 and XIAP by majorly targeting CDK9. Contrastively, it was relatively low toxic to the bone marrow cells of healthy donors. In MV4-11 xenograft mouse models, oral administration of CDKI-73 resulted in a marked inhibition of tumor growth (p < 0.0001) and prolongation of animal life span (P < 0.001) without causing body weight loss and other overt toxicities. The study suggests that CDKI-73 can be developed as a highly efficacious and orally deliverable therapeutic agent for treatment of AML.

Clinical characteristics and prognosis of 34 cases of acute myeloid leukemia with FLT3 internal tandem duplication and MLL gene rearrangement

目的探讨同时伴FLT3-ITD突变及MLL基因异常的急性髓系白血病（AML）患者的临床特征及转归。方法回顾性分析34例同时伴FLT3-ITD突变及MLL基因异常的AML患者的临床资料，比较化疗、化疗加靶向药物治疗及allo-HSCT的疗效及影响因素。结果34例同时伴FLT3-ITD突变及MLL基因异常的AML患者占同期住院AML患者的2.02%。入院时WBC>30×109/L的患者占63.6%，其中WBC>50×109/L者占39.4%。FAB亚型中以M5比例最高，占35.3%，染色体核型异常者达63.6%，其中复杂异常占12.1%。34例患者中仅有FLT3-ITD及MLL基因异常（双基因异常）者11例（32.4%），具FLT3及MLL以外的1种及1种以上的基因异常（多基因异常）者23例（67.6%）。34例患者2个疗程完全缓解（CR）率为29.4%，7例（20.6%）化疗≥3个疗程后CR，CR患者的早期复发率为52.9%。WBC>50×109/L以及多基因异常的患者2个疗程CR率较低（7.7%、5.4%），其中具有3种以上基因异常的患者无一例CR。34例患者2年总生存（OS）率为28.8%（95%CI 13.5%~46.0%），2年无病生存（DFS）率为27.1%（95% CI 12.5%~44.0%）。18例仅使用化疗或化疗加靶向药物治疗的患者，17例在2年内死亡，1例放弃治疗后失访。接受allo-HSCT治疗的患者3年OS率为43.4%（95%CI 13.7%~70.4%），3年DFS率为42.7%（95% CI 13.4%~69.7%）。结论同时伴FLT3-ITD突变及MLL基因异常的AML患者FAB分型以M5多见，常伴高白细胞血症、细胞遗传学异常及多基因异常。患者化疗缓解率低，早期复发率高，长期生存率低。高白细胞血症、多基因异常可能是此类患者疗效差的重要原因，allo-HSCT可改善患者的转归。

Set4 is a chromatin-associated protein, promotes survival during oxidative stress, and regulates stress response genes in yeast

The Set4 protein in the yeast Saccharomyces cerevisiae contains both a PHD finger and a SET domain, a common signature of chromatin-associated proteins, and shares sequence homology with the yeast protein Set3, the fly protein UpSET, and the human protein mixed-lineage leukemia 5 (MLL5). However, the biological role for Set4 and its potential function in chromatin regulation has not been well defined. Here, we analyzed yeast cell phenotypes associated with loss of Set4 or its overexpression, which revealed that Set4 protects against oxidative stress induced by hydrogen peroxide. Gene expression analysis indicated that Set4 promotes the activation of stress response genes in the presence of oxidative insults. Using ChIP analysis and other biochemical assays, we also found that Set4 interacts with chromatin and directly localizes to stress response genes upon oxidative stress. However, recombinant Set4 did not show detectable methyltransferase activity on histones. Our findings also suggest that Set4 abundance in the cell is balanced under normal and stress conditions to promote survival. Overall, these results suggest a model in which Set4 is a stress-responsive, chromatin-associated protein that activates gene expression programs required for cellular protection against oxidative stress. This work advances our understanding of mechanisms that protect cells during oxidative stress and further defines the role of the Set3-Set4 subfamily of SET domain-containing proteins in controlling gene expression in response to adverse environmental conditions.

MLL Gene Rearranged Acute Lymphoblastic Leukemia

MLL Gene Rearranged Acute Lymphoblastic Leukemia