MLL Leukemia Research Articles

Role of Epigenetic Readers in Pathogenesis and Therapy of Acute Leukemias

Acute myeloid leukemia (AML) is an aggressive hematological malignancy with a dismal survival rate of less than 30 percent and is thus a disease where novel therapeutics are urgently required. AML is highly heterogeneous at both the molecular and clinical level. However, recurrent themes of altered transcriptional and epigenetic control are evident from sequencing and mechanistic studies. Recently, critical epigenetic regulators have surfaced as mediators of leukemogenesis and potential therapeutic targets in AML and other hematological malignancies. We and others have recently demonstrated that the Bromodomain and Extra Terminal (BET) protein family of transcriptional adapter proteins are required for transformation by MLL-fusion proteins, where they facilitate the localization of aberrant transcriptional complexes to chromatin at genes critical for leukemogenesis. Furthermore, we and others have demonstrated that small molecule inhibitors, such as I-BET151 and JQ1, of the protein-protein interactions between these proteins and acetylated lysine residues in histone tails are effective in pre-clinical models of MLL leukemias. We have recently extended studies of BET inhibition to other forms of AML using I-BET151 and found a range of sensitivities across a panel of AML cell lines and patient samples with common recurrent mutations. These cell lines also demonstrate a common cellular response to MLL-fusion leukemias, with induction of apoptosis and G1/0 arrest, suggesting that similar transcriptional programs may be targeted. In further investigating this, we identified a common transcriptional program downregulated in sensitive leukemia subtypes. In addition, using diagnostic gene expression profiles, we also demonstrate that this program can classify AML patients, suggesting that the program may provide predictive biomarkers and potentially identify those patients most likely to respond to BET inhibitions. Focusing on the most common mutation in AML, mutations of the nucleophosmin gene (NPM1c mutations), we present evidence to suggest that wild-type NPM1 has an inhibitory influence on BRD4, which is relieved upon NPM1c mutation and cytosplasmic dislocation. NPM1c mutation allows upregulation of the core transcriptional program facilitating leukemia development, and this program is abrogated by I-BET therapy. Finally, we demonstrate the efficacy of I-BET151 in human cell lines, a unique murine model, and in primary patient samples of NPM1c AML. Taken together, we have used BET inhibitors as a laboratory tool to dissect out mechanisms of aberrant transcriptional control in AML. Furthermore our pre-clinical evaluation supports clinical trials of BET inhibitors across multiple AML subtypes and identifies potential predictive biomarkers of sensitivity and response to inform these studies. Disclosures:No relevant conflicts of interest to declare.

Modeling Chronic Myelomonocytic Leukemia Through Patient-Derived Induced Pluripotent Stem Cells

Chronic myelomonocytic leukemia (CMML), the most common entity among myelodysplastic syndrome/myeloproliferative neoplasm, is characterized by monocytosis, morphologic dysplasia, and progression to acute myeloid leukemia. Despite its relatively high incidence, the pathogenesis of CMML remains elusive, mainly due to the paucity of suitable animal models and the difficulties in the establishment of CMML cell lines. As reprogramming technology has been established as a new powerful tool for disease modeling, here we developed induced pluripotent stem cells (iPSC) from CMML leukemic cells. By introducing episomal vectors expressing OCT3/4, SOX2, KLF4, L-MYC, LIN28 combined with shRNA for p53, several lines of iPSC were generated from CD34+cells of a healthy donor (wild-type: WT) and a CMML patient with der (1; 7) (q10; p10) translocation. All iPSC expressed pluripotent surface markers (TRA1-60 and SSEA4) and stemness-related genes (NANOG, OCT3/4, SOX2, KLF4, C-MYC, and REL). The der (1; 7) (q10; p10) translocation were detected in all CMML iPSC.When co-cultured with 10T1/2 stromal cells in the presence of VEGF, CMML and WT iPSC generated a comparable frequency of CD34+ CD43+ hematopoietic progenitor cells (HPC). However, when cultured in cytokine-supplemented semisolid medium, CMML iPSC-derived CD34+ CD43+ HPC yielded an increased number of hematopoietic colonies with larger sizes, especially CFU-GM and CFU-GEMM, compared with WT iPSC-derived cells. Importantly, CMML iPSC-derived hematopoietic colonies mainly consisted of monoblasts with a high nucleus/cytoplasm ratio, while those of WT iPSC predominantly composed of macrophages. Flow cytometric analysis showed marked increases of CD34+ hematopoietic progenitors and CD13+ myeloid cells in CMML iPSC-derived hematopoietic colonies. Of note, among myeloid lineages, there were remarkable increases in CD14+ monocytic cells and CD24+ CD14- immature granulocytes, which were unique characteristics of human CMML. In addition, CMML iPSC-derived CD13+myeloid cells exhibited a weak expression of CD56, which was never detected in WT iPSC-derived cells, suggesting CMML iPSC-derived hematopoietic cells recapitulate the original phenotype of CMML. Notably, we found that CMML iPSC-derived HPC retained the ability to serially replate and generate colonies even after the fourth plating, although essentially no WT iPSC-derived colonies were detected after the second plating, suggesting enhanced self-renewal capacity of CMML iPSC-derived HPC. In addition, when cultured in methylcellulose without cytokines, CMML iPSC-derived HPC were able to form spontaneous hematopoietic colonies, in contrast to WT iPSC-derived cells that gave rise to almost no detectable colonies. Taken together, these results suggest CMML iPSC-derived HPC possess the multiple biologic properties of CMML leukemic blasts.Then, we conducted a comprehensive gene expression and DNA methylation profiling of WT and CMML parental CD34+ cells, iPSC, and iPSC-derived CD34+ CD43+ HPC. Strikingly, although gene expression and DNA methylation status were quite different between WT and CMML parental CD34+ cells (R2 = 0.72 for gene expression and 0.90 for DNA methylation), WT and CMML iPSC-derived HPC exhibited similar gene expression and DNA methylation pattern (R2= 0.92 and 0.96), indicating reprogramming followed by redifferentiation may enable to obtain more homogenous population of normal and CMML cells that reside in almost the same differentiation stage. Using these multi-omics platforms, we searched differentially expressed and methylated genes between WT and CMML iPSC-derived HPC to identify molecular abnormalities that contribute to the pathophysiology of CMML. Indeed, gene set enrichment analysis revealed that embryonic stem cell (ESC)-related gene sets were enriched in CMML iPSC-derived HPC, which is consistent with a previous report that showed murine MLL leukemia stem cells employ a transcriptional program shared with ESC. Functional analysis of candidate genes is underway to further define the molecular aberrations involved in the CMML pathogenesis.In summary, we have established a novel CMML model of patient-derived iPSC and revealed their derived HPC recapitulate the disease's primary features. These findings highlight the iPSC as an attractive platform to investigate the cellular and molecular pathophysiology of CMML. Disclosures:Kurokawa:Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Consultancy, Research Funding.

Enhancers of Polycomb EPC1 and EPC2 sustain the oncogenic potential of MLL leukemia stem cells.

Through a targeted knockdown (KD) screen of chromatin regulatory genes we identified the EP400 complex components EPC1 and EPC2 as critical oncogenic co-factors in acute myeloid leukemia (AML). EPC1 and EPC2 were required for the clonogenic potential of human AML cells of multiple molecular subtypes. Focusing on MLL-mutated AML as an exemplar, Epc1 or Epc2 KD induced apoptosis of murine MLL-AF9 AML cells and abolished leukemia stem cell potential. By contrast, normal hematopoietic stem and progenitor cells (HSPC) were spared. Similar selectivity was observed for human primary AML cells versus normal CD34+ HSPC. In keeping with these distinct functional consequences, Epc1 or Epc2 KD induced divergent transcriptional consequences in murine MLL-AF9 granulocyte-macrophage progenitor-like (GMP) cells versus normal GMP, with a signature of increased MYC activity in leukemic but not normal cells. This was caused by accumulation of MYC protein and was also observed following KD of other EP400 complex genes. Pharmacological inhibition of MYC:MAX dimerization, or concomitant MYC KD, reduced apoptosis following EPC1 KD, linking the accumulation of MYC to cell death. Therefore EPC1 and EPC2 are components of a complex which directly or indirectly serves to prevent MYC accumulation and AML cell apoptosis, thus sustaining oncogenic potential.

Epigenetic Roles of MLL Oncoproteins Are Dependent on NF-κB

MLL fusion proteins in leukemia induce aberrant transcriptional elongation and associated chromatin perturbations; however, the upstream signaling pathways and activators that recruit or retain MLL oncoproteins at initiated promoters are unknown. Through functional and comparative genomic studies, we identified an essential role for NF-κB signaling in MLL leukemia. Suppression of NF-κB led to robust antileukemia effects that phenocopied loss of functional MLL oncoprotein or associated epigenetic cofactors. The NF-κB subunit RELA occupies promoter regions of crucial MLL target genes and sustains the MLL-dependent leukemia stem cell program. IKK/NF-κB signaling is required for wild-type and fusion MLL protein retention and maintenance of associated histone modifications, providing a molecular rationale for enhanced efficacy in therapeutic targeting of this pathway in MLL leukemias.

Linking MLL Leukemia with Integrin Signaling

Identification of tractable signaling molecules essential for leukemogenesis facilitates the development of effective targeted therapies. In this issue of Cancer Cell, Miller and colleagues report that Integrin Beta 3, which is largely dispensable for normal hematopoiesis, plays an important role and is a potential therapeutic target in mixed lineage leukemia.

RE: Acute myeloid leukemia associated with FGFR1 abnormalities

We have read with great interest the article entitled ‘Acute myeloid leukemia associated with FGFR1 abnormalities’ by Lee et al. [1]. As mentioned, clonal eosinophilic disorders related with abnormalities of PDGFRA, PDGFRB, and FGFR1 are a newly emphasized entity in the ‘2008 WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues’ [2]. The myeloid and lymphoid neoplasms with eosinophilia and FGFR1 abnormality, previously known as ‘8p11 myeloproliferative syndrome (EMS),’ [3] is a disease with poor prognosis, which presents in various forms of hematologic malignancies and requires rigorous, close clinical monitoring after diagnosing this disease form, as previous reports of EMS describe rapidly changing disease courses under certain circumstances [4]. The authors mention multiple partner genes involved in these FGFR1 rearrangements, including ZNF198, CEP110, and others, in which a FGFR1 break-apart FISH analysis can be a remarkably useful tool for its diagnosis. Resemblance to the diagnosis of MLL leukemia, FISH analysis is very suitable for the detection of involvement of the main gene (MLL, and in this case the FGFR1 gene), although cannot specify the many different partner genes. To our knowledge, more than 30 MLL partner genes have been newly diagnosed over the last 8 years using long distance inverse polymerase chain reaction (LDI-PCR) performed at Diagnostic Center of Acute Leukemia (DCAL) at Frankfurt, Germany [5]. We have also applied the method and detected ZMYM2– FGFR1 [6, 7] and CEP110–FGFR1 rearrangement using genomic DNA samples (unpublished data). We suggest that as for the cases with MLL rearrangement, FGFR1 rearrangements could benefit from the precise identification of their corresponding partner genes, if possible with genomic DNA samples using methods of long distance (LD)or LDI-PCR. As described for case 2 of Lee et al. [1], our case with a ZMYM2–FGFR1 rearrangement also displayed a submicroscopic deletion of the FGFR1 gene, as was demonstrated by microarray studies [6]. This may argue for the fact that the commonly deleted regions may have contained genes with an adverse effect on leukemogenesis in these two patients. Therefore, it would be quite useful to have characterized these regions in more detail to evaluate the prognostic impact of these commonly deleted regions. Notably, FGFR1 rearrangements (or 8p11 involvement) have been studied in only 19 Asian patients including our case series [4, 6, 8], indicating that this type of genetic rearrangement is rather sporadic. We suggest the need for international collaborative studies among distinct ethnic entities (e.g., Asian ethnicity) to further determine the clinical, laboratory, and molecular-genetic characteristics of this orphan disease entity of FGFR1 rearrangements. J. J. Yang Department of Medicine, Graduate School, Kyung Hee University, Seoul, Korea

Mechanisms of Mixed-Lineage Leukemia

Advances in our understanding of the genetic determinants of leukemia have translated to better treatment options and improved survival of patients with acute myeloid and acute lymphoid leukemia. However, some leukemias, such as those bearing 11q23 (MLL) translocations, result in aggressive diseases with a relatively poor prognosis, despite improved treatments such as allogeneic hematopoietic stem cell transplantation. This article will briefly review the functions and regulation of wild-type MLL during normal hematopoiesis, while focusing on recent advances in our understanding of the molecular mechanisms governing MLL leukemias. The transcriptional targets, cooperating signaling pathways and molecular machinery involved in MLL-associated leukemias will be discussed, as well as how these may be harnessed for more personalized treatment of this disease.

Synthesis, activity and metabolic stability of non-ribose containing inhibitors of histone methyltransferase DOT1L

Histone methyltransferase DOT1L is a drug target for MLL leukemia. We report an efficient synthesis of a cyclopentane-containing compound that potently and selectively inhibits DOT1L (Ki = 1.1 nM) as well as H3K79 methylation (IC50 ~ 200 nM). Importantly, this compound exhibits a high stability in plasma and liver microsomes, suggesting it is a better drug candidate.

Enhancers of Polycomb EPC1 and EPC2 Prevent Accumulation of MYC to Sustain the Oncogenic Potential of MLL Leukemia Stem Cells

Abstract 400Dysregulation of the structure and function of chromatin is common in myeloid malignancy. We used a lentiviral screening strategy to target for knockdown ∼270 genes coding for chromatin regulatory proteins in human THP-1 AML cells. THP-1 cells harbor a t(9;11) translocation which is the cytogenetic hallmark of MLL-AF9, found in ∼5% of patients with AML. The strategy identified the EP400 chromatin complex components EPC1 and EPC2 as critical oncogenic co-factors in MLL leukaemia. Knockdown of both EPC1 and EPC2 in human MLL-mutated cell lines and of Epc1 and Epc2 in experimentally initiated murine MLL-AF9 AML cells led to loss of AML cell clonogenic potential, due to induction of apoptosis. The phenotype was rescued by forced expression of human EPC1 or EPC2 in Epc knockdown cells. Epc1 and Epc2 knockdown MLL-AF9 AML cells failed to initiate AML upon syngeneic transplantation into secondary murine recipients, while control cells initiated short latency leukemia. Likewise, xenogeneic transplantation of EPC1 or EPC2 knockdown primary human MLL-AF9 AML failed to engraft recipients, while control cells which engrafted every recipient to variable extent. In complete contrast, knockdown of EPC1 or EPC2 did not adversely impact on either the in vitro clonogenic or in vivo repopulating potential of primary murine KIT+ or human CD34+ normal haematopoietic stem and progenitor cells. These data demonstrate a remarkable selective dependency of MLL-mutated leukemia stem cells, but not normal hematopoietic stem and progenitor cells, on Epc1 and Epc2.EPC1 and EPC2 are part of an EP400 centred complex with a role in the DNA damage response. The complex is also known to associate with MYC. Consistent with a role for EPC containing complexes in the DNA damage response, Epc1 and Epc2 KD in both normal and leukemic cells induced γH2A.X. However, only in leukemia cells was a transcriptional signature of MYC activation noted together with accumulation of MYC protein, prior to induction of Bim and subsequent apoptosis. This observation reveals a novel mechanism operational in leukaemia cells but not normal myeloid cells by which protein levels of the critical cellular oncogene MYC are limited by EPC/EP400. This counterintuitive observation refines the paradigm for MYC in transformed cells: when the function of EPC and its associated EP400 complex components is inhibited, thus impairing the DNA damage response, MYC accumulates and sensitises cells to apoptotic death. Thus EPC1 and EPC2 are components of a complex which serves to prevent MYC accumulation in myeloid leukemia cells and their sensitisation to apoptosis, thus sustaining oncogenic potential. Therapeutic targeting of this complex may facilitate restoration of the natural tumour suppressor mechanisms that prevent cellular transformation by MYC and enhance chemosensitivity of AML cells. Disclosures:No relevant conflicts of interest to declare.

Abstract 4869: Whole genome sequence analysis of MLL rearranged infant acute lymphoblastic leukemias reveals remarkably few somatic mutations: A Report From the St Jude Children's Research Hospital - Washington University Pediatric Cancer Genome Project

Abstract Infant acute lymphoblastic leukemia (ALL) is characterized by MLL rearrangements (MLLr) and poor prognosis. To determine the complement of somatic mutations in this high risk leukemia, we performed whole genome sequencing (WGS) on 22 infants with MLL rearranged ALL. An analysis of the structure of the MLLr revealed that over half had complex rearrangements involving either three or more chromosomes, carried cryptic rearrangements, or contained at the breakpoints deletions, amplifications, insertions, or inversion of sequences. In three cases, genetic rearrangements were predicted to generate in addition to the MLL-partner gene fusion, novel in-frame fusions including KRAS-MLL; RAD51B-MLL / AFF1-RAD51B, AFF1-RAD51B-MLL; MLLT10-ATP5L-YPEL4 / ATP5L-YPEL4. An analysis of the number of non-silent mutations revealed infant ALL to have the lowest frequency of somatic mutations of any cancer sequenced to date. After removal of SVs and CNAs associated with the MLLr, a mean of only 3.5 SVs and 2.2 SNVs affecting the coding region of annotated genes or regulatory RNAs were detected per case. Despite the paucity of mutations several pathways were recurrently targeted including PI3K/RAS pathway in 45% (KRAS (n=4), NRAS (n=2), and non-recurrent mutations in NF1, PTPN11, PIK3R1, and ARHGAP32 (p200Rho/GAP)), B cell differentiation in 23% as a result of mono-allelic deletion or gains of PAX5, 14% with deletions of the CDKN2A/B, and 2 cases with focal deletions of the non-coding RNA genes DLEU1/2. WGS of two infant ALL relapse samples and comparison with their matched diagnostic samples revealed a marked increase in the number of mutations at relapse. Moreover, an analysis of the allelic ratios of mutated genes revealed clonal heterogeneity at diagnosis with relapse appearing to arise from a minor diagnostic clone. Because of the exceedingly low number of mutations detected in infant ALL, we used exome sequencing to determine the frequency of non-silent SNVs in 20 MLLr leukemias (9 ALLs, 10 AMLs and 1 AUL) in older children (7-19 yrs). This analysis revealed that non-infant pediatric MLL leukemias harbor a significantly higher number of non-silent somatic SNVs than infant ALL (mean 7.4/case in older patients vs 2.2/case in infants, p<0.001). Although the higher frequency of mutations may be a reflection of age, the low number of cooperating mutations in infants raises the possibility that the target cell of transformation differs between infants and older children, with the cells present during early development requiring fewer cooperating mutations to induce leukemia. In summary our data demonstrated an exceedingly small number of mutations in infant leukemia. The number of detected somatic mutations may represent the lower limit required to transform a normal human cell into cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4869. doi:1538-7445.AM2012-4869

The Histone Demethylase KDM1A Sustains the Oncogenic Potential of MLL-AF9 Leukemia Stem Cells

Using a mouse model of human MLL-AF9 leukemia, we identified the lysine-specific demethylase KDM1A (LSD1 or AOF2) as an essential regulator of leukemia stem cell (LSC) potential. KDM1A acts at genomic loci bound by MLL-AF9 to sustain expression of the associated oncogenic program, thus preventing differentiation and apoptosis. Invitro and invivo pharmacologic targeting of KDM1A using tranylcypromine analogs active in the nanomolar range phenocopied Kdm1a knockdown in both murine and primary human AML cells exhibiting MLL translocations. By contrast, the clonogenic and repopulating potential of normal hematopoietic stem and progenitor cells was spared. Our data establish KDM1A as a key effector of the differentiation block in MLL leukemia, which may be selectively targeted to therapeutic effect.

Essential Role for PU.1 in MEIS1 Activation and MLL Fusion Leukemia,

Abstract 3466Down-regulation of transcription factor PU.1, a key regulator of hematopoiesis, induces myeloid leukemia in mice, demonstrating a role of PU.1 as tumor suppressor. Recent studies, however, have also suggested that PU.1 is required for repopulation/self-renewal capacity of normal hematopoietic stem cells (HSCs), and presence of PU.1 activity may be necessary to favor growth of myeloid leukemia stem cells. To explore whether PU.1 could possibly act as an oncogene in the development of certain type of myeloid leukemia, we set to look for differential up-regulation of PU.1 among AML patients with distinct cytogenetic and genetic alterations in public databases. Consistent with recent molecular studies showing suppression of PU.1 expression by AML1-ETO and PML-RARa fusion proteins, PU.1 expresses at a significant lower level in AML patients with t(8;21) and t(15;17) translocations. In contrast, PU.1 expression level in MLL leukemia patients is significantly higher than that of other subgroups of AML. In addition, we found that a set of PU.1 direct target genes, as defined by genome wide location analysis of this factor, expresses at higher level in MLL leukemia patients comparing with those with t(8;21) and t(15;17) translocations, supporting an increased PU.1 activity in this subgroup of leukemia. In our effort to characterize the functional consequence of high expression of PU.1 in AML, we found that PU.1 plays an essential role in activation of MEIS1, an oncogene essential for MLL leukemia stem cell potential, and in development of MLL fusion leukemia. MEIS1, as PU.1, is differentially up-regulated in MLL leukemia patients, and expresses at a significant lower level in AML patients with t(8;21) and t(15;17) translocations. Among AML patients with higher level MEIS1 expression, a positive correlation was observed between expression of PU.1 and that of MEIS1. Using promoter reporter assay, electro mobility shift assay (EMSA) and chromatin immunoprecipiation (ChIP) analysis, we found that PU.1 directly binds to and activates MEIS1 promoter in vitro and in vivo. Analysis of a hypomorphic PU.1 mouse model indicated that PU.1 is required to maintain Meis1 expression in murine HSCs and progenitors, and knockdown of PU.1 in patient-derived MLL leukemia cell lines resulted in lower enrichment of PU.1 protein at MEIS1 promoter, accompanied by down-regulation of MEIS1 expression and decreased proliferation and survival of these cells. We are now examining whether the ability of MLL-AF9 fusion protein to drive leukemia is compromised in PU.1-deficient mouse HSC/HPCs, and whether introduction of exogenous Meis1 can compensate for the loss of PU.1 in the development of MLL-AF9 leukemia in mouse bone marrow transplantation model. Finally, we are also testing knock-down of PU.1 as a therapeutic approach to primary AMLs isolated from MLL leukemia patients. Collectively, our data indicate that PU.1 is required for the pathogenesis of MLL associated leukemia, at least partially, through direct activation of MEIS1. In veiw of the dependency of MEIS1 in MLL leukemic transformation, targeting PU.1 mediated MEIS1 gene activation could be an alternative or synergistic approach for MLL leukemia therapies aimed at inhibition of DOT1L and HOXA9. Disclosures:No relevant conflicts of interest to declare.

Predicting Clinical Dose-Exposure and Exposure-Response Relationships of Pan-Antiapoptotic BCL-2 Family Inhibitor Obatoclax in MLL Rearranged Infant Leukemias From Preclinical Disease Models and Adult Experience

Abstract 2580Challenges in developing new agents for infant MLL leukemia are compounded by rarity of cases, inherent drug resistance and unique vulnerabilities of infants to treatment-related morbidity and mortality. An additional limitation is the extent to which preclinical data and adult clinical experience can predict clinical dose-exposure and exposure-response relationships and guide dosing strategies (dose, duration, combination, schedule) and study designs (sample size, sampling scheme). Pharmacometric approaches are increasingly being used to quantify drug action and clinical performance. These require a continuum of models with hypotheses and assumptions and pharmacologic rationale to be challenged by targeted experimentation and subsequent model refinement. Key assumptions and experimental conditions are reassessed to optimize experimental designs and test evolving understanding about drug behavior and disease biology. Here we establish “proof-of-concept” quantitative pharmacology relationships between data derived from in vitro obatoclax activity in cell lines and patient samples, historical adult experience and a preclinical diseased mouse model to optimize strategies to treat infant leukemia.Obatoclax in vitro activity was quantified by MTT assays in MLL-rearranged cell lines and primary infant ALL and bilineal MLL leukemia samples. Obatoclax biodistribution was characterized in MLL leukemia-bearing mice to evaluate its potential to achieve target exposures in infants with leukemia by the following steps: 1) A nonlinear mixed-effect model based on existing obatoclax toxicokinetic data in healthy Balb/C mice guided design of a pilot study in diseased NOD-scid-IL-2Rgnull (NSG) mice where target exposure requirements were based on the AUC24h associated with peak oligonucleosomal DNA release as an apoptosis biomarker in an adult Phase I CLL trial (O'Brien 2008) and single-agent MTT assays of obatoclax in MLL cell lines and primary infant leukemia samples. 2) NSG MLL-ENL bilineal leukemia bearing mice with established xenografts received a single IV bolus dose of obatoclax (1.2 or 4.8 mg/kg; 100% or 400% of predicted target exposure). 3) Obatoclax was measured in the plasma, spleen, liver, kidney and brain for 24 h after drug administration (courtesy GeminX). A physiologically-based pharmacokinetic model (PBPK) model was developed to predict exposures in mouse target tissues based on obatoclax physicochemical properties and then scaled to a human infant.Preclinical in vitro experiments suggested IC50 values between 13 and 918 nM in primary MLL leukemia cells. Population mean (% RSE) parameter estimates for plasma clearance (CL), intercompartmental clearance (Q), plasma distribution volume (Vd) and peripheral distribution volume (V2) were 5.1 (10.1) L/h/kg, 1.78 (37) L/h/kg, 9.15 (10.2) L/kg and 17.3 (23.8) L/kg, respectively. PK parameters were close to those predicted from healthy Balb/C mice except that Cmax was lower, possibly suggesting a larger Vd in mice with disease. The half-life was almost 4-fold higher suggesting slower elimination due to mouse strain or disease burden. There was sustained drug retention in spleen, liver, kidney and brain. Brain:plasma were ∼2-10:1, higher than the <1:1 typical of most drugs, indicating excellent CNS penetration. The PBPK model for obatoclax in the mouse compared well with observed biodistribution in diseased NSG mouse tissues (Fig 1). Though brain penetration was good, deviations between observed and PBPK-simulated exposure values may suggest involvement of transporters. PBPK simulation of obatoclax administration to a 1 yr, 10 kg human infant suggested that adequate drug exposure to target organs should be achievable at clinically relevant doses (Fig 2).This work links models that define obatoclax in vitro exposure targets with in vivo exposure obtained in leukemia bearing mice. Predictability of these models relative to infant leukemia will be assessed following completion of the ongoing COG phase I obatoclax trial in relapsed/refractory pediatric cancers (ADVL0816). Our analyses imply that projecting outcome in treating infant MLL disease with obatoclax will be possible once these models are further refined. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Pharmacological Inhibition or Genetic Knockdown of Kdm1a (Lsd1 or Aof2) Induces Differentiation of MLL Acute Myeloid Leukaemia Stem Cells

Abstract 58Histone demethylases are candidate therapeutic targets in cancer. We analysed a previously published microarray expression dataset of murine acute myeloid leukemias (AML) experimentally initiated with human MLL fusion oncogenes to identify histone demethylases whose expression correlated with the frequency of leukemia stem cells (LSCs). Only expression of Kdm1a correlated positively and significantly (p<0.001) with LSC frequency, a finding confirmed by quantitative PCR of selected leukemias. To investigate whether Kdm1a has a critical role in MLL LSCs, we targeted Kdm1a transcripts for knockdown in murine MLL-AF9 AML cells using lentivirally expressed shRNAs. Knockdown AML cells formed significantly fewer colonies in semi-solid culture compared with AML cells expressing a non-targeting control shRNA (12±7% of control; n=4; p=0.01), and the extent of Kdm1a knockdown correlated significantly (six shRNAs tested) with loss of AML cell colony forming (CFC) potential (p<0.005). Residual Kdm1a knockdown colonies predominantly exhibited “Type 2” morphology (i.e. containing macrophages), a finding confirmed by examination of cytospin preparations. The phenotype was rescued by forced expression of human KDM1A. Kdm1a knockdown MLL-AF9 AML cells were unable to initiate leukemia in secondary transplant assays, in contrast to control cells which readily induced short latency AML. These data demonstrate that Kdm1a is required to prevent differentiation of murine MLL-AF9 AML LSCs.Tranylcypromine (TCP) is a licensed monoamine oxidase inhibitor that inhibits KDM1A, with an IC50 of 5–10uM. Treatment of murine MLL-AF9 AML cells, or human THP1 MLL-AF9 cells, with TCP phenocopied Kdm1a knockdown at a dose close to the KDM1A IC50; both murine and human MLL-AF9 cells exhibited reduced proliferation, fewer colonies and increased macrophage differentiation. Next, we tested the effect of KDM1A knockdown or TCP treatment (25uM) on primary human MLL leukemia cells. KDM1A knockdown reduced proliferation, induced morphological differentiation and induced apoptosis in the two samples of MLL-AF6 leukemia tested (5–20 fold fewer cells remained after a ten day culture versus control cells). Likewise, TCP treatment reduced proliferation 4-fold over five days (MLL-AF6 n=2; MLL-AF10 n=1), blocked CFC formation, reduced the proportion of blasts and increased the proportion of cells with features of monocyte/macrophage differentiation. Of note, addition of 25uM TCP to normal murine KIT+ BM stem and progenitor cells cultured in methylcellulose did not significantly reduce CFC frequency or size or induce macrophage differentiation, suggesting that MLL LSCs are more sensitive than normal BM cells to a given level of KDM1A inhibition.To investigate the mechanism by which KDM1A inhibits a differentiation programme in MLL-AF9 LSCs, we performed exon array analysis of Kdm1a knockdown versus control LSCs 48 hours following lentiviral infection, prior to appearance of morphologically differentiated cells. Down regulated transcriptional regulators included Id2, Hmgb3 and Egr1, but not Hoxa, Meis1 or Myb. Up regulated transcriptional regulators included Irf8. Knockdown of Id2 in murine MLL-AF9 AML cells reduced CFC frequency. In contrast, forced expression of Id2 enhanced CFC frequency both of control MLL-AF9 AML cells and of Kdm1a knockdown LSCs, the latter representing a partial phenotypic rescue of Kdm1a knockdown.These data demonstrate that KDM1A prevents differentiation of MLL LSCs by regulating the relative expression of pro- versus anti-differentiation transcriptional regulators and highlight it as a novel therapeutic target in MLL leukemias. Disclosures:No relevant conflicts of interest to declare.

Comparing Human Fetal Liver, Cord Blood and Adult Bone Marrow Stem/Progenitor Hematopoietic Cells As Cells of Origin of Human MLL Leukemias

Abstract 2956Acute leukemias in newborns, children, adults and elderly share the same name, but their genetic, biology and prognosis are significantly different. These differences may be explained by many factors including the nature of the recurrent genetic abnormalities, tolerance to chemotherapy, co-morbidities, etc. One noteworthy potential difference is the cell of origin in which the initial genetic event occurs, which can be a fetal liver, cord blood or adult bone marrow stem/progenitor cell. Genetically engineered mouse models of leukemias and gene expression studies on human acute lymphoblastic leukemias (ALL) suggest that leukemias developing in utero (i.e. in an early ontogenic cell of origin) are biologically distinct from child and adult leukemias. However, direct comparison of human hematopoietic stem/progenitor cells at different stages of ontogeny has not been tested experimentally.Over 70% of infant acute leukemias and 5–10% of adult leukemias exhibit rearrangements of the mixed-lineage leukemia gene (MLL). Therefore, MLL translocations are highly relevant oncogenes to investigate the leukemic potential of the different cells of origin. The MLL-ENL (MLL-MLLT1) fusion gene, from the t(11;19) (q23;p13.3), is one of the most frequent MLL fusion and can occur in infants and adults. It is found in both acute myeloid leukemias (AML) and ALL (both B-cell and T-cell ALL) and we have previously shown that it generates human B-ALL in immunodeficient mice when introduced into lineage depleted human cord blood cells using a retrovirus (Barabé et al, Science 2007).The same experimental design was used to study the cells of origin; CD34+ cells from human fetal liver (FL), cord blood (CB) and adult bone marrow (BM) were infected with either a retrovirus encoding MLL-ENL and an enhanced green fluorescent protein (EGFP) marker gene, or a control retrovirus encoding EGFP only, then injected into sub-lethally irradiated immunodeficient mice. After transduction with MLL-ENL, FL cells generated leukemias in 8/10 mice, CB cells in 6/6 mice and adult BM cells in only 2/7 mice, although four times more cells were transduced and injected. Adult BM cells were very difficult to infect with retroviruses and were clearly not as potent to reconstitute a human graft in immunodeficient mice in comparison to CB cells. All generated leukemias were B-ALL, regardless of the cell of origin. The leukemic phenotype (CD19+, HLA-DR+, CD20-, IgM-, IgG-, CD34 variable) was similar in all three cell types. Mice injected with MLL-ENL-transduced FL and adult BM cells that were not leukemic had a normal human graft but no EGFP cells. The frequency of leukemia-initiating cells (L-IC) was evaluated at 0.2–1% by limiting dilution in secondary mice and no significant differences were observed between the three cell types. Leukemic cells were harvested from the BM of primary mice and cultured in vitro under conditions supportive of both B-lymphoid and myeloid cells. All leukemias were able to grow in vitro and at least one culture derived from each cell type (FL, CB and adult BM) switched to a myeloid phenotype (monocytic morphology with CD33+ and loss of CD19), demonstrating that leukemias derived from all 3 cell types still have myeloid potential.Thus, our results suggest that MLL-ENL can generate leukemia in FL, CB and adult BM CD34+ cells. The lower penetrance of leukemia with adult BM cells is probably due to the difficulty to infect these cells and their limited reconstitution ability. MLL-ENL induces a leukemic program in all three cell types with similar clinical features, cell surface phenotype, frequency of L-IC and conserved bi-lineage potential (B and myeloid). Gene expression profiling and clonality studies are currently performed on leukemias generated from the 3 different cell types. So far, no significant differences between FL, CB and adult BM as cells of origin of MLL leukemias have been identified, suggesting that the nature of the oncogenic hit is probably more important than the ontogenic stage of the cell of origin. Disclosures:No relevant conflicts of interest to declare.

TTC5, a TPR Motif Cofactor Protein Required for Myeloid Leukaemogenesis

Abstract 59Identification of genes and cellular pathways that are critical for the function of leukemia stem cells (LSC) could lead to novel therapeutic approaches. We present data which demonstrates that TTC5 (also called Strap) is required in acute leukaemogenesis. In a targeted lentiviral shRNA knockdown (KD) screen of chromatin regulatory factors, we identified the EP300 co-factor TTC5 as required for survival of human THP-1 MLL-AF9 acute monoblastic leukaemia cells. TTC5 is a tetratricopeptide repeat (TPR) motif protein known to be a stress responsive co-factor and activator of the histone acetyltransferase EP300. Using two separate shRNAs targeting different parts of the transcript, TTC5 KD in THP-1 cells resulted in G1 cell cycle arrest and the initiation of myeloid differentiation (as evidenced by down regulation of KIT/CD117 and up regulation of the monocyte differentiation marker CD36 over 48–72 hours) prior to apoptosis. Colony formation in semi-solid culture was completely abolished. TTC5 KD also induced apoptosis and loss of colony forming potential in other myeloid leukaemia cell lines including Fujioka, Kasumi-1, NB4, HL60, K562 and Mono-Mac-1. In xenotransplantation assays, TTC5 KD THP-1 cells failed to initiate AML in contract to control cells infected with a non-targeting control (NTC) hairpin, which induced short latency AML (range 51–72 days). Likewise, lentivirally infected TTC5 KD primary human acute lymphoblastic leukaemia cells from a patient with a t(4;11) translocation, the cytogenetic hallmark of MLL-AF4, failed to induce lethal leukemia when xenografted into neonatal mice, in contrast to NTC cells which induced lethal ALL in recipients of median latency 117 days. Separately, TTC5 KD induced proliferative arrest, differentiation and death in in vitro analyses of primary human AML cells which exhibited MLL associated translocations or trisomy 11 (frequently associated with a partial tandem duplication of MLL) (n=4 patients). These data suggest a critical role for TTC5 in human leukaemic haematopoiesis.To confirm our conclusions, we extended our investigations in a murine model of human leukaemic haematopoiesis initiated by MLL-AF9. TTC5 KD MLL-AF9 AML cells formed significantly fewer colonies in methylcellulose versus control cells and failed to initiate AML in secondary transplantation experiments versus control cells which induced short latency disease (48 days).To investigate the mechanism by which TTC5 knockdown initiates the cell cycle arrest and myeloid differentiation phenotype observed in THP-1 cells, we performed exon array analyses 48 hours following lentiviral infection. Gene set enrichment analysis (GSEA) demonstrated co-ordinate down regulation of an MLL leukemia stem cell maintenance program as well as genes bound by the transcription factor MYB. In contrast there was co-ordinate up regulation of genes known to be positively regulated by PU.1 or IRF8, two myeloid transcription factors that promote differentiation. These significant transcriptional changes occurred in the absence of change in the levels of MYB or PU.1 transcripts, arguing that TTC5 may be critical in regulating the function of these transcription factors rather than modulating their expression. Published data from the FANTOM4 consortium identified MYB and, to a lesser extent, PU.1 as key transcriptional regulators of CD36 expression (Suzuki et al., 2008, Nature Genetics). We confirmed up regulation of CD36 upon MYB knockdown in THP-1 cells. CD36 up regulation was not observed when either MYC or EP300 was knocked down, suggesting that TTC5 is required for MYB function and loss of TTC5 perturbs MYB activity, initiating a differentiation program. The mechanism by which TTC5 regulates MYB activity is under investigation but appears independent of EP300. We propose that in addition to the role of TTC5 in the stress response, it is a critical cofactor protein that regulates haematopoietic transcription factors and is required for human leukaemogenesis. Disclosures:No relevant conflicts of interest to declare.

Activity of the Novel Ribonucleotide Reductase Inhibitor Didox on Human and Murine Models of Acute Leukemia,

Abstract 3637Acute Myeloid Leukemia (AML) is an aggressive, genetically heterogeneous disease wherein immature myeloid progenitors crowd out the normal hematopoietic cells leading to marrow failure and ultimately death. Each year in the United States there are approximately 12,000 new cases and 9,000 deaths from AML. Combination chemotherapy of an anthracycline and cytarabine has remained the mainstay of frontline treatment despite decades of active research. Treatment will often lead to a remission; however, the majority of patients relapse with chemoresistant disease. This highlights the need for the development of new therapies for patients. The Ribonucleotide Reductase (RR) reaction facilitates the conversion of ribonucleotides to deoxyribonucleotides via a free radical intermediate and is the rate-limiting step in DNA replication. The anti-tumor activity of Hydroxyurea is due to its ability to quench the free radical intermediate generated at the M2 subunit of RR ultimately resulting in a depletion of the cell's deoxyribonucleotide pool. This chemotherapeutic is useful for the palliative treatment of AML; however, its myelosuppressive effects are dose limiting. 3,4-Dihydroxybenzohydroamic acid (Didox) is a RR inhibitor with a mechanism of action similar to that of Hydroxyurea that has been shown to be less myelosuppressive in an animal model. Didox has been examined for toxicity in Phase I and Phase II trials in the United Kingdom in several malignancies. These clinical trials have reported Didox to be well tolerated at 6 g/m2. Major dose limiting organ toxicities were hepatic and renal. Given this low toxicity and mechanism of action we tested the activity of Didox in pilot studies of human and murine leukemias.In vitro studies were done to assess the Inhibitory Concentration (IC50) against five human AML cell lines, KG1a, OCI-AML3, THP-1, HL60 and K562. Cells were exposed to a titration of Didox for 72 hours and viability determined. The average IC50 of Didox was 5.76 μM (Range 3.62–7.37 μM).Initial In vivo studies were carried out in a syngeniec, therapy resistant MLL-ENL driven AML model. Murine leukemias were injected into sublethally irradiated recipients and allowed to engraft. Animals were given five days of Didox 425 mg/kg or a control treatment via intraperitoneal injection. Response and survival data were collected. A nimals tolerated the therapy well. Treated animals demonstrated a significant survival benefit in two separately derived MLL leukemias (one expressing the Flt3-ITD and one expressing NRasG12D) with p values of 0.0094 and 0.009 respectively. To determine the efficacy of Didox against a murine Acute Lymphoblastic Leukemia (ALL) model we injected syngeniec Balb/c mice with Baf3 cells engineered to express the p210 BCR-ABL fusion protein. We found that while the animals tolerated Didox, treatment did not provide a survival benefit, p =0.3581. This suggests that the use of Didox would be more efficacious in the treatment of AML than in ALL.Didox is a novel ribonucleotide reductase inhibitor with IC50 values in the low micromolar range for a panel of human and murine cell lines. Pilot studies in our syngeneic mouse models show that Didox is well tolerated in AML and ALL. Moreover, Didox demonstrates a survival benefit in chemoresistant murine AMLs. This data would imply Didox has activity in AML and additional studies to further characterize its activity are underway. Disclosures:Elford:Molecules for Health: Equity Ownership, Patents & Royalties.

Abstract B119: AF4 mimetic peptide SPK111 prolongs survival of MLL xenografted leukemias.

Abstract Acute leukemias with balanced translocations of the MLL gene are aggressive blood cancers with generally poor prognosis. Importantly, MLL leukemias are the most common type of leukemia in babies and account for 70% of the secondary leukemias that arise following treatment with topoisomerase II inhibitors. Only 40% of infants treated for acute lymphoblastic leukemia (ALL) with MLL gene rearrangements survive to the age of five while the survival rate is 90% for patients in the same age group whose disease lacks a translocated MLL gene. Hence, better treatment options for MLL-rearranged leukemias need to be explored. As results of reciprocal translocations, the 5' coding region of MLL is fused to the 3' coding region of one of many different genes. This results in transcripts that encode chimeric proteins. AF4 and AF9 proteins are the two most frequent fusion partners of MLL. They are components of the protein complex P-TEFb that promotes transcriptional elongation. Misdirection of the transcriptional elongation complex is one proposed mechanism by which oncogenic transformation occurs in MLL leukemias. Our laboratory previously identified the amino acids required for direct physical interaction between AF4 and AF9 and designed an AF4 mimetic peptide that disrupts the AF4 - AF9 interaction. Treatment of cell lines with MLL-AF4 and MLL-AF9 translocations with the mimetic peptide leads to necrotic cell death without affecting normal hematopoietic cells. In order to further establish the AF4 - AF9 interaction as an effective target for MLL leukemias, we tested effect of a modified peptide designed to enhance its stability in xenografted mice. The peptide, designated SPK-111 exherts effective antitumor activity without causing overt toxicity. It significantly improves the survival of NOD/SCID mice engrafted with human cell lines carrying MLL gene rearrangements when used as a single agent. Here, we describe the in vivo activity of the peptide and propose that it, or other derivatives, may represent a clinically important agent for the treatment of MLL leukemias. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B119.

The Pathogenesis of Mixed-Lineage Leukemia

Aggressive leukemias arise in both children and adults as a result of rearrangements to the mixed-lineage leukemia gene (MLL) located on chromosome 11q23. MLL encodes a large histone methyltransferase that directly binds DNA and positively regulates gene transcription, including homeobox (HOX) genes. MLL is involved in chromosomal translocations, partial tandem duplications, and amplifications, all of which result in hematopoietic malignancies due to sustained HOX expression and stalled differentiation. MLL lesions are associated with both acute myeloid leukemia and acute lymphoid leukemia and are usually associated with a relatively poor prognosis despite improved treatment options such as allogeneic hematopoietic stem cell transplantation, which underscores the need for new treatment regimens. Recent advances have begun to reveal the molecular mechanisms that drive MLL-associated leukemias, which, in turn, have provided opportunities for therapeutic development. Here, we discuss the etiology of MLL leukemias and potential directions for future therapy.

Abstract 278: Role of AF4 and Dot1 in MLL-AF9 mediated leukemogenesis

Abstract Mixed lineage leukemia (MLL) is characterized by rearrangement of the MLL gene at chromosome band 11q23. MLL gene rearrangements give rise to chimeric proteins that contain the N-terminal portion of MLL fused to the C-terminal portion of over 50 different fusion partners. MLL chimeras do not retain the histone methyltransferase activity of wild type MLL and it is unclear how they deregulate the expression of MLL target genes. Our laboratory studies the two most common MLL fusion partners encountered in patients with leukemia, AF4 and AF9. We have previously shown that AF4 and AF9 heterodimerize and peptides that block this interaction are toxic to MLL leukemia cell lines. This supports the hypothesis that the interaction of MLL-AF9 with AF4 is required for leukemogenesis. However, we have also shown that AF4 and H3K79 methyltransferase, Dot1 have similar binding sites on AF9. Furthermore, we have found that Dot1 is required for immortalization of hematopoietic progenitor cells by MLL fusion genes. The work of Yokoyama and others demonstrated that AF4, ENL (a close homologue of AF9) and Dot1L do not exist in the same multimeric complex. To understand the role of AF4 and Dot1 in MLL-AF9 mediated leukemogenesis, we made point mutants of AF9 with differential binding for AF4 and Dot1. Based on our preliminary data and recent studies, we hypothesize that the binding of AF4 and not Dot1 to MLL-AF9 is required for MLL-AF9 mediated leukemogenesis. Nevertheless, Dot1 remains a critical factor in MLL-AF9 leukemia. We hypothesize that Dot1 recruitment is mediated by wild type AF9 rather than MLL-AF9. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 278. doi:10.1158/1538-7445.AM2011-278