IntroductionEpigenetic modification is an important mechanism for the regulation of transcription and maintenance of cell identity with cell division. Hence, many histone-modifying enzymes have been identified as crucial for maintaining normal hematopoietic stem cells (HSCs) as well as leukemia-initiating cells (LICs). Disruptor of telomeric silencing 1 (Dot1) is a novel class of histone methyltransferase (HMT) that was first identified in yeast for its ability to dysregulate gene silencing near telomeres. 1-3 Dot1 and its mammalian homolog, Dot1l (Dot1-like), is currently the only identified histone 3 lysine 79 (H3K79) methyltransferase. 4,5 Since its discovery, many studies have shown an essential role for Dot1l and H3K79 methylation in embryonic development, prenatal hematopoiesis, and leukemia. 6,7 Recently, Dot1l has been shown to be specifically required for transformation by Mixed Lineage Leukemia (MLL) fusion proteins. 8 However, the role of Dot1l in normal postnatal hematopoiesis has not been definitely shown.In general, Dot1l and H3K79 methylation is associated with transcriptional activation. [9][10][11] Interestingly, the loss of Dot1l seems to regulate a relatively short list of genes instead of globally downregulating the entire transcriptome. This effect has been particularly noted in the context of embryonic stem cells, suggesting a specific biologic role for Dot1l. 12 Further studies using constitutive Dot1l knockout mouse models provide additional evidence for a role of Dot1l in stem cell biology. Analyses of Dot1l knockout embryonic stem cells and yolk sac cells show that the loss of Dot1l leads to cell cycle defects, chromosomal aberrations, and prenatal hematopoietic abnormalities. 13,14 However, the 2 constitutive Dot1l knockout mouse lines are embryonic lethal by E10.5 and E13.5, precluding the examination of Dot1l loss in postnatal mice and in definitive hematopoiesis.Dot1l is strongly associated with leukemias arising from translocations of the MLL gene, which fuse MLL in frame to more than 60 different translocation partners. 15 Multiple studies show that Dot1l interacts with many of the most common MLL translocation partners, such as AF9, ENL, AF4, and AF10, in a complex promoting transcriptional elongation. [16][17][18][19][20][21] Recent studies also show that the reciprocal translocation protein AF4-MLL can interact with Dot1l while exhibiting different gene expression profiles from conventional MLL translocation proteins. 21,22 For this paper, MLL translocation refers to the fusion of N-terminus of MLL with a translocation partner that can associate with Dot1l. Additional evidence from MLL translocation-containing cell lines and patient samples shows up-regulation of H3K79 methylation at MLL translocation target genes, including HOXA9 and MEIS1, which are critical for leukemogenesis. 19,23 Furthermore, the loss of H3K79 methylation through knockdown of Dot1l leads to reduced expression of these genes. 19,23 However, it is not clear whether Dot1l is required for MLL translocation-m...

• The PAFc subunit, Cdc73, is required for the proliferation and proper epigenetic regulation of proleukemic oncogenes in AML cells.• Disrupting the MLL-PAFc interaction selectively inhibits the growth of MLL-associated leukemic cells without altering normal hematopoietic stem cell function.MLL rearrangements are common in leukemia and considered an adverse risk factor. Through interactions with the polymerase-associated factor complex (PAFc), mixed lineage leukemia (MLL) fusion proteins activate genes critical for blocking differentiation, such as HOXA9. Here we investigate whether the MLL-PAFc interaction can be exploited therapeutically using both genetic and biochemical approaches. We tested the genetic requirement of the PAFc in acute myeloid leukemia (AML) using a conditional allele of the PAFc subunit, Cdc73. We show that the PAFc is indiscriminately necessary for the proliferation of AML cells through the epigenetic regulation of proleukemogenic target genes, such as MEIS1 and Bcl2. To investigate the therapeutic potential of targeting the MLL-PAFc interaction, we engineered a dominant negative fragment of MLL capable of binding to the PAFc. Disruption of the MLL-PAFc interaction selectively inhibits the proliferation of MLL leukemic cells without affecting cells transformed by an unrelated E2A-HLF fusion protein. Using in vivo hematopoietic reconstitution assays, we demonstrate that disruption of the MLL-PAFc does not alter normal hematopoietic stem cell function. Together, our data show a selective growth inhibition of MLL-associated leukemic cells and tolerance of normal hematopoiesis to disruption of the MLL-PAFc interaction establishing the MLL-PAFc interaction as an attractive therapeutic target. (Blood. 2013;122(11):1914-1922

TP53 is a key tumor suppressor gene that plays an important role in regulating apoptosis, senescence, and DNA damage repair in response to cellular stress. Although somewhat rare, TP53 -mutated AML has been identified as an important molecular subgroup with a prognosis that is arguably the worst of any. Survival beyond one year is rare after induction chemotherapy with or without consolidative allogeneic stem cell transplant. Although response rates have been improved with hypomethylating agents, outcomes remain particularly poor due to short response duration. Improvements in our understanding of AML genetics and biology have led to a surge in novel treatment options, though the clinical applicability of these agents in TP53 -mutated disease remains largely unknown. This review will focus on the epidemiology, molecular characteristics, and clinical significance of TP53 mutations in AML as well as emerging treatment options that are currently being studied.

56 Balanced chromosomal translocations of the MLL gene located on chromosome 11q23 result in the expression of a chimeric fusion proteins with enhanced transcriptional activity. The HOX genes and their co-factors, such as MEIS1 and PBX2, are critical downstream targets of MLL fusion proteins and essential for transformation. Previously we showed MLL fusion proteins are critically dependent on a direct interaction with the RNA Pol II Associated Factor complex (PAFc). PAFc is a protein complex important for the initiation, elongation and termination of transcription. It is also necessary for histone H2B K120 mono-ubiquitination through the direct recruitment of the BRE1/RAD6 E3 ubiquitin ligase complex. MLL fusion proteins make two direct contacts with the PAF1 and CTR9 subunits of the PAFc that are crucial for MLL fusion protein mediated transformation. Deletion of regions of MLL that interact with PAFc abrogates AML in mouse bone marrow transplantation assays. Here we tested the general requirement for PAFc in AML using a conditional knockout mouse model of one component of PAFc, Cdc73. These studies show that PAFc is necessary for growth of both E2A-HLF and MLL-AF9 transformed cells. Excision of Cdc73 leads to decreased expression of the MLL target genes Hoxa9 and Meis1, decreased colony formation and decreased proliferation of leukemic blasts and ultimately apoptosis. We then performed chromatin immunoprecipitation assays to assess the binding of PAFc and MLL to target loci with and without Cdc73. Excision of Cdc73 leads to a rapid decrease in association of PAFc as well as MLL fusion proteins and wild type MLL at target loci confirming that proper targeting of MLL fusion proteins requires PAFc. A decrease in H3K4me3 and H2Bub is also observed and consistent with a role of PAFc in the deposition of these epigenetic marks. We then sought to disrupt the MLL-PAFc interaction through expression of a small 40 amino acid fragment of MLL that interacts with the PAF1 subunit of PAFc. As the MLL-PAFc interaction involves interactions between MLL and both CTR9 and PAF1, it was unknown whether targeting one interaction site would be sufficient to disrupt transformation. Indeed, expression of the short fragment encompassing the pre-CxxC region of MLL acts as a dominant negative and disrupts the MLL-PAFc interaction, significantly decreasing Hox gene expression, colony formation and cell proliferation of MLL-AF9 transformed cells. Importantly, expression of the MLL fragment selectively inhibited MLL fusion mediated leukemic transformation and cell growth while the growth and proliferation of E2A-HLF cells is unaffected. Together these data show that targeting the MLL-PAFc interaction with a small MLL fragment can act as a dominant negative and selectively inhibit the growth of AML cells transformed with MLL fusion proteins. These data also suggest the MLL-PAF1 interaction surface is a promising region for therapeutic targeting. Disclosures: No relevant conflicts of interest to declare.

3291 Histone modifying enzymes are crucial regulators of hematopoiesis that are commonly disrupted in acute leukemia. DOT1L has emerged as a particularly important methyltransferase in leukemias with Mixed Lineage Leukemia (MLL) rearrangements. Leukemogenic MLL fusion proteins transform primarily through upregulation of A-cluster HOX genes, including HOXA9 and the HOX cofactor MEIS1. Many of the most common MLL translocation partners including the AF4 family members, AF9, ENL, and AF10, form the Elongation Assisting Proteins (EAP) complex that includes DOT1L. DOT1L is the only known histone methyltransferase that methylates histone H3 on lysine 79 (H3K79). Increasing evidence suggests this histone modification, which is generally associated with transcriptional activation, is essential for MLL fusion protein mediated oncogenicity. Chromatin immunoprecipitation (ChIP) on MLL fusion protein containing cell lines shows higher levels of H3K79 methylation across the HOXA9 and MEIS1 loci compared to non-MLL fusion protein containing cell lines. Similarly, patient samples with MLL fusion proteins show elevated H3K79 methylation. Finally, knockdown of DOT1L has been shown to inhibit growth of MLL rearranged cell lines. These findings suggest that DOT1L may be an effective therapeutic target, however further development of DOT1L inhibitors will be dependent on assessing the efficacy of DOT1L disruption in a wider range of leukemic cells as well as determining the potential toxicity and effect on normal hematopoiesis. Given that constitutive Dot1l knock out is early embryonic lethal, we established conditional Dot1l knockout mouse from gene trap sperm obtained from the Knock Out Mouse Project (KOMP). Dot1l targeted animals were crossed to CreER animals so that Dot1l knock out could be induced with 4-OHT or tamoxifen. To determine the effects of Dot1l deletion in transformation ability, mouse were injected with 5-flurouracil and bone marrow cells were transduced with retrovirus expressing oncogenes in the presence or absence of 4-OHT and growth in methocult media was examined. These experiments showed that growth of cells transformed by MLL-AF9 is completely abolished by Dot1l deletion while transformations by HOXA9/MEIS1 (downstream targets of MLL fusion proteins) and E2A-HLF (which expresses very low levels of HOXA9/MEIS1 and transform through other mechanisms) were unaffected. We also examined the toxicity of Dot1l deletion in vivo by treating mice with tamoxifen and monitoring survival and assessing hematopoiesis. Immunohistochemical studies show that loss of Dot1l is associated with widespread loss of lysine 79 di- and tri-methylation in organs including liver, spleen, bone marrow, testis, muscle and gastrointestinal track with some residual methylation retained in the central nervous system. Immunophenotypic analysis of mouse bone marrow 3–4 weeks after tamoxifen treatment revealed reduction in HSCs, GMPs, MEPs, and CLPs with Dot1l excision. Bone marrow transplantations and cell cycle experiments are currently underway to further characterize the hematopoietic defects in Dot1l deficient animals. Additional experiments will be needed to determine if Dot1l activity is required in other leukemias with high level HOX expression. Together these results suggest that Dot1l is a promising therapeutic target as it is specifically required for transformation by MLL fusion leukemia, however bone marrow suppression occurs with Dot1l inhibition. Disclosures: No relevant conflicts of interest to declare.