ALBERT

Description: Mixed Lineage Leukemia gene rearrangements (MLL-r) account for nearly 10% of human acute leukemia cases and are generally associated with poor prognosis. Previous studies have revealed an essential role of the histone H3K79 methyltransferase Disruptor of Telomeric Silencing-1 Like (DOT1L) in MLL-r leukemogenesis. Our recent report (Chen et al. 2015 Nature Medicine) further identified a role for histone acetylation in DOT1L dependent gene expression driven by MLL-fusion proteins including MEIS1 and HOXA cluster genes. A first-in-human Phase I clinical trial demonstrated clinical activity of DOT1L inhibition in MLL-r leukemia patients, thus providing a potential opportunity for treating these malignant diseases. Nevertheless, the incomplete silencing of the leukemic program by only targeting DOT1L motivates the need for additional and perhaps combinational approaches to improve therapies against MLL-r leukemias. To enhance the efficacy of DOT1L inhibition, we sought to identify genes whose suppression would synergize with the DOT1L inhibitors to suppress the proliferation of mouse bone marrow progenitors transformed with MLL-AF9. We conducted a pooled RNAi screen using a customized library composed of 2,252 shRNA targeting 468 epigenetic regulators (i.e. writers, readers, and erasers of chromatin modifications; Fig 1). The integrated shRNA sequences were assessed using high-throughput sequencing. By comparing the change in frequency of each shRNA construct cultured in control vs. an IC50 DOT1L inhibitor EPZ4777, we identified several candidate modulators of DOT1L dependency, which had multiple shRNAs selectivity depleted only in the DOT1L suppressed condition. Notably, using a network correlation study, we found that one of the top candidate genes Plant Homeodomain Finger Protein 20 (PHF20) is highly associated with histone acetylation in the mammalian epigenome. Knockdown of PHF20 drastically increased the sensitivity of MLL-AF9 leukemic blasts to DOT1L inhibitors through enhanced myeloid differentiation and reduced cell proliferation, colony formation, and re-plating capacity. Similar phenotypes were also observed in PHF20-deficient MLL-AF9 cells generated by CRISPR/Cas9-mediated gene knockout. PHF20 is an epigenetic adaptor protein that has no predicted enzymatic activity. To investigate the role of PHF20, we conducted a CRISPR functional domain screen and identified the requirement of the chromatin reader domains in PHF20, including the Tudor domains and the PHD-finger, in supporting the survival of MLL-r leukemic cells upon DOT1L inhibition. We also performed RNA-seq and found that suppression of PHF20 facilitated the silencing of the MLL-AF9 leukemic program induced by DOT1L inhibitor treatment. Chromatin immunoprecipitation and sequencing (ChIP-seq) analyses validated that PHF20 contributes to the maintenance of histone acetylation including H3K9ac and H4K16ac at MLL-AF9 target loci. In line with the profound loss of histone acetylation at MLL-AF9 target loci in PHF20-depleted cells, we found that knockdown of a known PHF20 interacting partner KAT8 (a histone acetyltransferase; also known as MOF or MYST1) phenocopies the effects observed in PHF20-knockdown cells. Finally, we showed that pharmacological inhibition of DOT1L and KAT8 synergistically suppresses the proliferation and survival of MLL-AF9 leukemic cells. These data collectively highlight the involvement of a novel DOT1L-PHF20-KAT8 axis in mammalian gene regulation and MLL-r leukemogenesis. In summary, our studies show that MLL-rearrangements may drive leukemic transformation by coordinating an epigenetic network involving several histone modifications associated with gene transcription (e.g. H3K79 methylation and H3K9/H4K16 acetylation). Our results also suggest that simultaneous targeting of multiple components of this epigenetic feed-forward loop including DOT1L and PHF20/KAT8 may provide a novel and more effective approach against MLL-r leukemia. Disclosures Bradner: Novartis Institutes for BioMedical Research: Employment. Armstrong:Epizyme, Inc: Consultancy; Vitae Pharmaceuticals: Consultancy; Imago Biosciences: Consultancy; Janssen Pharmaceutical: Consultancy.

Description: To identify cooperating lesions in core-binding factor acute myeloid leukemia, we performed single-nucleotide polymorphism-array analysis on 300 diagnostic and 41 relapse adult and pediatric leukemia samples. We identified a mean of 1.28 copy number alterations per case at diagnosis in both patient populations. Recurrent minimally deleted regions (MDRs) were identified at 7q36.1 (7.7%), 9q21.32 (5%), 11p13 (2.3%), and 17q11.2 (2%). Approximately one-half of the 7q deletions were detectable only by single-nucleotide polymorphism-array analysis because of their limited size. Sequence analysis of MLL3, contained within the 7q36.1 MDR, in 46 diagnostic samples revealed one truncating mutation in a leukemia lacking a 7q deletion. Recurrent focal gains were identified at 8q24.21 (4.7%) and 11q25 (1.7%), both containing a single noncoding RNA. Recurrent regions of copy-neutral loss-of-heterozygosity were identified at 1p (1%), 4q (0.7%), and 19p (0.7%), with known mutated cancer genes present in the minimally altered region of 1p (NRAS) and 4q (TET2). Analysis of relapse samples identified recurrent MDRs at 3q13.31 (12.2%), 5q (4.9%), and 17p (4.9%), with the 3q13.31 region containing only LSAMP, a putative tumor suppressor. Determining the role of these lesions in leukemogenesis and drug resistance should provide important insights into core-binding factor acute myeloid leukemia.

Description: The interaction of Menin (MEN1) and MLL (MLL1, KMT2A) is a dependency and potential therapeutic opportunity against NPM1-mutant (NPM1mut) and MLL-rearranged (MLL-r) leukemias. Concomitant activating driver mutations in the gene encoding the tyrosine kinase FLT3 occur in both leukemias and are particularly common in the NPM1mut subtype. Transcriptional profiling upon pharmacological inhibition of the Menin-MLL complex revealed specific changes in gene expression with downregulation of the MEIS1 transcription-factor and its transcriptional target gene FLT3 being most pronounced. Combining Menin-MLL-inhibition with specific small-molecule kinase inhibitors of FLT3-phosphorylation resulted in a significantly superior reduction of phosphorylated FLT3 and transcriptional suppression of genes downstream to FLT3 signaling. The drug combination induced synergistic inhibition of proliferation as well as enhanced apoptosis and differentiation compared to single-drug treatment in models of human and murine NPM1mut and MLL-r leukemias harboring an FLT3 mutation. Primary AML cells harvested from patients with NPM1mutFLT3mut AML showed significantly better responses to combined Menin and FLT3-inhibition than to single-drug or vehicle control treatment, while AML cells with wildtype NPM1, MLL, and FLT3 were not affected by any of the two drugs. In vivo treatment of leukemic animals with MLL-r FLT3mut leukemia reduced leukemia burden significantly and prolonged survival compared to the single-drug and vehicle control groups. Our data suggest that combined Menin-MLL and FLT3-inhibition represents a novel and promising therapeutic strategy for patients with NPM1mut or MLL-r leukemia and concurrent FLT3 mutation.

Description: Acute myeloid leukemia (AML) is characterized by molecular heterogeneity. As commonly altered genomic regions point to candidate genes involved in leukemogenesis, we used microarray-based comparative genomic hybridization and single nucleotide polymorphism profiling data of 391 AML cases to further narrow down genomic regions of interest. Targeted resequencing of 1000 genes located in the critical regions was performed in a representative cohort of 50 AML samples comprising all major cytogenetic subgroups. We identified 120 missense/nonsense mutations as well as 60 insertions/deletions affecting 73 different genes (∼ 3.6 tumor-specific aberrations/AML). While most of the newly identified alterations were nonrecurrent, we observed an enrichment of mutations affecting genes involved in epigenetic regulation including known candidates like TET2, TET1, DNMT3A, and DNMT1, as well as mutations in the histone methyltransferases NSD1, EZH2, and MLL3. Furthermore, we found mutations in the splicing factor SFPQ and in the nonclassic regulators of mRNA processing CTCF and RAD21. These splicing-related mutations affected 10% of AML patients in a mutually exclusive manner. In conclusion, we could identify a large number of alterations in genes involved in aberrant splicing and epigenetic regulation in genomic regions commonly altered in AML, highlighting their important role in the molecular pathogenesis of AML.

Description: NPM1mutant (NPM1mut) and MLL1-rearranged (MLL-r) acute myeloid leukemias (AMLs) exhibit aberrant expression of HOX and MEIS1 transcription factors and commonly harbor an activating mutation in the receptor tyrosine kinase FLT3. Pharmacologic inhibition of the menin-MLL1 complex reverses leukemogenic gene expression including MEIS1 and FLT3 and represents a therapeutic opportunity for the treatment of these leukemias. Here, we investigate the contribution of the menin-MLL1 complex to leukemic FLT3 signaling and assess the therapeutic potential of dual menin-MLL1 and FLT3 targeting. First, we performed RNA sequencing to delineate transcriptional changes associated with menin-MLL1 inhibition (-i) using the small molecule inhibitor MI503 in NPM1mut and MLL-r AMLs (OCI-AML3 and MV411 cells). In both leukemias, we confirmed MEIS1 and its target gene FLT3 to be among the most significantly downregulated genes. These results were validated in several human cell lines of NPM1mut or MLL-r AMLs and in a genetically engineered murine AML model harboring an NPM1mut and an internal tandem duplication mutation in the FLT3 gene (Npm1mut/+Flt3ITD/+,now referred to as Npm1mutFlt3ITD). Allele-specific qPCR upon MI503 treatment confirmed profound suppression of the FLT3ITD allele present in the MLL-r MV411 and MOLM13 cells. Total FLT3 protein expression was also reduced upon MI503 treatment in all tested NPM1mut and MLL-r AMLs. Next, we assessed the therapeutic potential of combined menin-MLL1-i and FLT3-i in the FLT3-ITD positive MLL-r and NPM1mut leukemias. We found dramatic synergistic growth inhibition and substantially enhanced apoptosis when combining MI503 with the specific small molecule FLT3 inhibitor AC220 (Quizartinib) compared to monotreatment or vehicle control in the FLT3ITD positive MLL-r MV4-11 and MOLM-13 cells. Similar results were obtained when MI503 was combined with other specific FLT3 inhibitors - Crenolanib and Gilteritinib. As the murine Npm1mutFlt3ITD AML cells harbor the Flt3ITD F692L gatekeeper mutation that conveys drug resistance to most FLT3 inhibitors, we used Ponatinib as a combination partner for MI503 in these cells and found similar synergistic suppression of proliferation and colony formation. No drug sensitivity to single or combo treatment was observed in the human HL60 and NB4 cells, or the murine Hoxa9-Meis1 transformed cells (all wildtype for NPM1, MLL, FLT3). To investigate the mechanism of drug synergy we then performed immunoblotting of phosphorylated (activated) FLT3 (pFLT3). As expected, we observed reduced pFLT3 upon direct FLT3 inhibition with AC220 and found decreased total FLT3 and pFLT3 with MI503 monotreatment. Of note, we observed the most pronounced pFLT3 reduction with combo treatment, most likely reflecting the cooperative effect of direct pFLT3 inhibition with AC220 and transcriptional suppression of total FLT3 via MI503. Transcriptional profiling revealed most dramatic reduction of FLT3 downstream signature gene expression including MYC and MYC-dependent genes upon combinatorial treatment compared to single drug or vehicle controls. Ectopic expression of Meis1 or Hoxa9-Meis1 in the Npm1mutFlt3ITD cells led to increased Flt3 gene and protein expression and partially rescued the anti-proliferative effect of MI503 and combined menin-MLL1-i and FLT3-i. Next, we assessed the combo drug regimen in a cell line derived leukemic xenograft murine model in vivo. We found significantly reduced leukemia burden defined by bone marrow engraftment after 14 days of treatment within the combo versus the single drug or vehicle control animals. In a separate experiment, animals that had been treated with the drug combo in vivo had a dramatically enhanced survival compared to the control groups. We then evaluated single and combo drug effects on five primary human NPM1mutFLT3ITD AML patient samples in a human stroma cell co-culture model. The most profound anti-leukemic effect was again detected with the combo treatment compared to all other controls. In summary, we demonstrate synergistic on-target activity against mutant FLT3 signaling with combined menin-MLL1 and FLT3 inhibition in leukemias with NPM1mut or MLL-r in vitro and in vivo. This concept may represent a novel therapeutic opportunity against these AMLs harboring a prognostically adverse FLT3-ITD. Disclosures Vassiliou: Kymab Ltd: Consultancy, Other: Minor Stockholder; Oxstem Ltd: Consultancy; Celgene: Research Funding. Armstrong:Mana Therapeutics: Consultancy, Equity Ownership; Accent Therapeutics: Consultancy, Equity Ownership; OxStem Oncology: Consultancy, Equity Ownership; Syros Pharmaceuticals: Consultancy, Equity Ownership; C4 Therapeutics: Consultancy, Equity Ownership; Cyteir Therapeutics: Consultancy, Equity Ownership; Janssen: Research Funding; Novartis: Research Funding; AstraZeneca: Research Funding; Epizyme, Inc.: Consultancy, Equity Ownership; Imago Biosciences, Inc.: Consultancy, Equity Ownership. Kühn:Pfizer: Consultancy; ABBVIE: Consultancy; Daiichi Sankyo: Other: travel Support; Celgene: Other: travel support.

Description: Genetic alterations of the mixed-lineage leukemia (MLL) gene are commonly identified in acute leukemias. In acute myeloid leukemia (AML), a partial tandem duplication (PTD) of MLL occurs in about 5-10% of AML patients and is associated with adverse prognosis. The mutation leads to an in-frame duplication of exons 5 to 11 resulting in the production of an aberrant MLL protein. Unlike chromosomal rearrangements of MLL, this mutation does not affect the functional histone 3 lysine 4 (H3K4) methyltransferase domain. However, AMLs carrying a MLL-PTD and MLL-rearranged leukemias share some common characteristics, such as overexpression of HOXA-cluster genes and dysregulated epigenetic functions. Recently, leukemias with various MLL-translocations have been shown to be dependent on the histone 3 lysine 79 (H3K79) methyltransferase, DOT1L, and are sensitive to EPZ004777, a recently described selective small-molecule DOT1L inhibitor. EPZ-5676, a DOT1L-inhibitor with improved potency and drug-like properties, has recently been identified and is currently under clinical investigation. To evaluate the therapeutic potential of DOT1L-inhibition in MLL-PTD positive leukemia cells, we assessed the effect of EPZ004777 on the MLL-PTD containing leukemia cell lines MUTZ-11 and EOL-1. In vitro treatment with EPZ004777 over a 14-day period resulted in dramatic reduction of cell proliferation compared to DMSO vehicle control in both cell lines beginning 7 days after the start of treatment. Similar results were obtained for MOLM-13, a leukemia cell line harboring a MLL-translocation, but not for HL-60, a non-MLL-rearranged leukemia cell line. To further investigate whether these findings reflect a selective response to EPZ004777 or non-specific drug toxicity, we first explored the genome-wide H3K79 dimethylation (H3K79me2) profile using chromatin immunoprecipitation (ChIP) followed by next generation sequencing in untreated MUTZ-11 cells. Across the HOXA-cluster locus, we detected a similar H3K79me2 distribution pattern as previously reported in MLL-rearranged leukemias. Further analysis of H3K79me2 in MUTZ-11 and EOL-1 cells after treatment with the inhibitor showed profound suppression of those marks as assessed by western blot and ChIP-PCR. Subsequent global gene expression analysis revealed concurrent downregulation of HOXA-cluster and other MLL-target genes after 7 days of DOT1L inhibition. To investigate the effect of EPZ004777 on the MLL-PTD positive cells in more detail, we analyzed cell differentiation and apoptosis upon a 10-day exposure to the compound. As previously described for EPZ004777-sensitive MLL-rearranged leukemias, drug treatment resulted in increased expression of CD11b and morphological changes consistent with myeloid cell differentiation. We further observed apoptotic cell death after EPZ004777 treatment as measured by an increase in the percentage of Annexin V positive cells and cleaved Caspase 3 protein compared to vehicle controls. In order to determine the effect of DOT1L inhibition in vivo, we tested the recently identified DOT1L-inhibitor, EPZ-5676, for its ability to inhibit leukemia growth in a subcutaneous EOL-1 xenograft model in immunocompromised rats. Similar to what we observed in vitro, continuous intravenous administration over 21 days led to substantial dose-dependent inhibition of tumor growth, abrogation of H3K79me2, and concurrent downregulation of selected MLL-target genes. Thus, we demonstrate unexpected sensitivity of MLL-PTD containing leukemia cell lines to the DOT1L inhibitors EPZ004777 in vitro and EPZ-5676 in vivo. These data suggest that patients with myeloid malignancies carrying this particular mutation might benefit from treatment with therapeutic approaches that target DOT1L. Disclosures: Daigle: Epizyme, Inc: Employment, Equity Ownership. Olhava:Epizyme Inc.: Employment. Pollock:Epizyme Inc.: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Armstrong:Epizyme Inc.: Has consulted for Epizyme Inc. Other.

Description: Key Points Relapsed AML with NPM1 mutation is genetically related to the primary leukemia and characterized by an increase in high-risk aberrations. DNMT3A mutations show the highest stability and thus may precede NPM1 mutations.

Description: To identify genomic alterations in chronic lymphocytic leukemia (CLL), we performed single-nucleotide polymorphism–array analysis using Affymetrix Version 6.0 on 353 samples from untreated patients entered in the CLL8 treatment trial. Based on paired-sample analysis (n = 144), a mean of 1.8 copy number alterations per patient were identified; approximately 60% of patients carried no copy number alterations other than those detected by fluorescence in situ hybridization analysis. Copy-neutral loss-of-heterozygosity was detected in 6% of CLL patients and was found most frequently on 13q, 17p, and 11q. Minimally deleted regions were refined on 13q14 (deleted in 61% of patients) to the DLEU1 and DLEU2 genes, on 11q22.3 (27% of patients) to ATM, on 2p16.1-2p15 (gained in 7% of patients) to a 1.9-Mb fragment containing 9 genes, and on 8q24.21 (5% of patients) to a segment 486 kb proximal to the MYC locus. 13q deletions exhibited proximal and distal breakpoint cluster regions. Among the most common novel lesions were deletions at 15q15.1 (4% of patients), with the smallest deletion (70.48 kb) found in the MGA locus. Sequence analysis of MGA in 59 samples revealed a truncating mutation in one CLL patient lacking a 15q deletion. MNT at 17p13.3, which in addition to MGA and MYC encodes for the network of MAX-interacting proteins, was also deleted recurrently.