MLL-r Leukemia Research Articles

Abstract 1175: Inhibition of NAMPT as a novel therapeutic strategy for infant leukemia

Abstract Novel targeted therapies are urgently needed for infant leukemia as this disease is highly aggressive and refractory to treatment resulting in poor survival rates. The NAD (Nicotinamide Adenine Dinucleotide) producing enzyme NAMPT (Nicotinamide Phosphoribosyltransferase) has been considered an attractive selective target for anti-cancer therapy due to the high dependency of tumor cells on NAD for energy metabolism and activity of NAD-dependent enzymes such as poly-ADP-ribose polymerases (PARPs) and sirtuins that play key roles in cancer cell survival. A newly developed NAMPT inhibitor, OT-82, was initially isolated for its high selective toxicity against a panel of adult leukemia cell lines. Here we investigated NAMPT inhibition as a therapeutic strategy for infant leukemias characterized by rearrangement of the MLL gene (MLL-r), by testing the potency of OT-82 in a panel of preclinical in vitro and in vivo models of MLL-r leukemia that are based on the use of patient-derived xenograft (PDX) cells. OT-82, as a single agent, dramatically reduced the viability of all tested MLL-r leukemia cell lines (n=9) and MLL-r leukemia PDX (n=6) with IC50s ranging from 0.15 to 3.82nM. While the IC50 for OT-82 correlated significantly with the IC50 of other NAMPT inhibitors STF-118804 and FK866, OT-82 was the most potent compound. When combining OT-82 with chemotherapeutic agents currently used to treat infants with leukemia, we observed significant synergy between OT-82 and cytarabine indicating the potential of OT-82 for chemosensitization. Consistent with NAMPT inhibition, OT-82 reduced cytosolic NAD+ levels in MLL-r leukemia cells and inhibited the activity of the NAD-requiring enzymes PARP-1 and SIRT-1, as exemplified by a decrease in PARylated PARP-1 levels and a p53-mediated increase in p21 levels, leading to apoptosis induction. Interestingly, despite the remarkable potency of OT-82 in killing MLL-r leukemia cells, a 25-fold difference in IC50 levels was noted across the cell line panel, with those lines harboring the MLL translocations most prevalent in infants, namely t(4;11) and t(11;19) translocations, being the most sensitive to the compound. A positive correlation was noted between baseline NAMPT mRNA levels and OT-82 IC50 (r=0.7712, P=0.015). Further in vivo testing of OT-82 showed impressive efficacy of the compound in MLL-r leukemia PDX-based animal models (n=6). OT-82 (p.o. 3x week, 40-50 mg/kg, for 3 or 6 weeks) was well tolerated and significantly delayed leukemia progression in 6/6 MLL-r leukemia xenografts with 5/6 achieving objective responses. OT-82 given as monotherapy was as effective as the routinely used triple combination treatment of vincristine, L-asparaginase and dexamethasone. Overall, these results demonstrate that NAMPT inhibition using OT-82 is highly effective against MLL-r leukemia and when combined with current chemotherapies may offer a more selective and potent therapeutic option for infants suffering from this disease. Citation Format: Klaartje Somers, Shiloh Middlemiss, Asel Biktasova, Mawar Karsa, Leanna Cheung, Angelika Kosciolek, Kathryn Evans, Chelsea Mayoh, Ursula R. Kees, Lioubov Korotchkina, Olga B. Chernova, Richard B. Lock, Andrei V. Gudkov, Michelle Haber, Murray D. Norris, Michelle J. Henderson. Inhibition of NAMPT as a novel therapeutic strategy for infant leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1175. doi:10.1158/1538-7445.AM2017-1175

Activating Mutations Are Potent Pro-Leukemic Mediators in Murine MLL-MLLT3 Leukemia That Cause Distinct Transcriptional Profiles

Acquired activating mutations in kinase/PI3K/RAS signaling pathways occur in about half of pediatric acute leukemia cases with Mixed Lineage Leukemiagene rearrangements (MLL-R; MLL, also known as KMT2A). Mutations resulting in activated signaling cooperate with the MLL-R in mouse leukemia models, however, detailed insight on the effect on the transcriptional and proteomic landscape is lacking. In infant MLL-R acute lymphoblastic leukemia (ALL), a subtype with a very poor prognosis, a majority of the activating mutations are subclonal, but the biological mechanisms by which subclonal mutations affect leukemogenesis remain unclear.Here we show that NRASG12D, FLT3internal tandem duplication(ITD)and FLT3N676K,cooperates with MLL-MLLT3 in myeloid leukemogenesis using a competitive murine retroviral bone marrow transplantation model. The addition of an activating mutation remodels the gene expression patterns of the MLL-R leukemia with distinct profiles for each mutation as determined by RNA-sequencing. Gene set enrichment analysis revealed enrichment of genes involved in chromatin assembly, transcription and stemness in leukemia induced by MLL-MLLT3 and NRASG12D, FLT3ITD or FLT3N676K. Leukemia induced by only MLL-MLLT3 displayed upregulation of genes involved in signal transduction suggesting activation of such pathways by alternative mechanisms. Upon secondary transplantation, mice receiving MLL-MLLT3-only leukemic cells succumbed to disease at a similar latency to those receiving MLL-MLLT3 and an activating mutation, supporting that fully transformed MLL-MLLT3 leukemias have sustained active signaling via high expression of genes involved in signal transduction.Using the same model as above but with significantly reduced numbers of transplanted cells containing an activating mutation, we could further show that a subclonal mutation, exemplified by FLT3N676K, causes significantly reduced disease latency as compared to mice receiving MLL-MLLT3 only (34 vs 50 days). The size of the double positive (MLL-MLLT3+FLT3N676K) and single positive (MLL-MLLT3) clones within each mouse was determined by flow cytometry revealing that 8/24 mice had a double positive subclone (≤50%). The clonal evolution of the double positive cells from 22/24 mice was assessed in secondary recipients showing three distinct patterns 1) increase in size, 19/22 mice 2) maintained, 2/22 mice or 3) decreased in size, 1/22 mice. Targeted gene re-sequencing of the latter leukemia that lost its MLLT3+FLT3N676K subclone, identified a de-novo CblA308T in the SH2-like domain, in the MLL-MLLT3 onlycells that had gained clonal dominance in the secondary recipient.The decreased disease latency in mice with subclonal activating mutations raises the possibility that cells with an activating mutation support the growth of other leukemic cells by direct cell-cell contact or through secreted factors. Transcriptome and proteomic analyses identified a high expression of the macrophage inhibitory factor (MIF), a pro-inflammatory cytokine, in mice with MLL-MLLT3 and NRASG12D, FLT3ITD or FLT3N676K. Addition of rMIF increased the survival of MLL-MLLT3 murine leukemic cells in vitro. Although additional factors likely mediate pro-leukemic effects in vivo, our data suggest that MIF could be one of these factors.In summary, our data demonstrate that activating mutations cooperate with the MLL-R in murine leukemogenesis and cause widespread changes in the transcriptional landscape. In addition, our results suggest that cells containing an activating mutation in addition to an MLL-fusion positively influence the survival and likely also the growth of other leukemic cells, suggesting a pro-leukemic effect mediated by interclonal cooperation between clones carrying distinct mutational set-ups in leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

The IRX1/HOXA connection: insights into a novel t(4;11)- specific cancer mechanism.

One hallmark of MLL-r leukemia is the highly specific gene expression signature indicative for commonly deregulated target genes. An usual read-out for this transcriptional deregulation is the HOXA gene cluster, where upregulated HOXA genes are detected in MLL-r AML and ALL patients. In case of t(4;11) leukemia, this simple picture becomes challenged, because these patients separate into HOXAhi- and HOXAlo-patients. HOXAlo-patients showed a reduced HOXA gene transcription, but instead overexpressed the homeobox gene IRX1. This transcriptional pattern was associated with a higher relapse rate and worse outcome. Here, we demonstrate that IRX1 binds to the MLL-AF4 complex at target gene promotors and counteract its promotor activating function. In addition, IRX1 induces transcription of HOXB4 and EGR family members. HOXB4 is usually a downstream target of c-KIT, WNT and TPO signaling pathways and necessary for maintaining and expanding in hematopoietic stem cells. EGR proteins control a p21-dependent quiescence program for hematopoietic stem cells. Both IRX1-dependend actions may help t(4;11) leukemia cells to establish a stem cell compartment. We also demonstrate that HDACi administration is functionally interfering with IRX1 and MLL-AF4, a finding which could help to improve new treatment options for t(4;11) patients.

Abstract C12: Identification of biomarkers and pathways associated with response to the DOT1L inhibitor Pinometostat (EPZ-5676) in MLL-r leukemia

Abstract Pinometostat is a highly selective first in class DOT1L inhibitor currently in Phase 1 clinical trials in adult and pediatric leukemia patients (pts) with MLL rearrangements (MLL-r or MLL-PTD). Preliminary results of the adult trial have demonstrated clinical activity including complete remissions in a subset of patients (Stein, 2014). Investigation and identification of candidate molecular correlates of pinometostat response in both pt samples and cell lines are reported. RNA and DNA were isolated from PBMCs and/or bone marrow collected prior to treatment from 18 pts enrolled in the adult pinometostat Phase 1 study (CT.gov: NCT01684150), at the following doses 24 (n = 2), 36 (n = 3), 54 (n = 6), 80 (n = 3) and 90 mg/m2/day doses (n = 4). mRNA transcript abundance was assessed using whole genome RNASeq and DNA variants were determined using a 194 gene panel, MyAML (Genection Inc.). Correlations of transcript abundance and DNA variants detected with categorical (responder = CR or PR [n = 3], or no response [n = 16]) and continuous response parameters (time on study [TOS], mean = 59 days: range = 8-196 days) were performed. For cell lines, whole genome RNASeq data was generated from 14 cell lines (MLL-r or MLL-PTD) with a range of in vitro sensitivity to pinometostat (cell proliferation IC50 2 nM to > 10 μM) and RNA transcripts identified as correlated with IC50 were submitted for pathway analysis. Univariate analyses revealed no DNA variants to be associated with either response category (FDR adjusted P < 0.05), however, a statistical association (unadjusted P = 0.05) was identified between increased TOS and those pts harboring t(11:19). Indicating that specific MLL fusion partners may elicit differential sensitivity to pinometostat therapy (2/2 CR pts had t(11:19)). Analysis of the baseline PBMC RNASeq data revealed 201 genes as significantly correlated with TOS (FDR adjusted P<0.05), these data were further analyzed using the Selventa causal modelling platform to identify pathways associated with TOS. Increased activity in pathways leading to differentiation, oxidative stress, inflammation, ras activation and decreased activities of PPAR-γ, DNA methylation and stem cell renewal were associated with increased TOS. Independent analysis of the cell line response data identified pathways that significantly overlapped with those observed in pt samples, with 4/7 of the pt derived mechanisms identified (PPAR-γ activity, inflammation, stem cell renewal and ras activation). In all cases directionality of pathway activity relative to pinometostat sensitivity were concordant between pt and cell line data. Based on these results the effect of a PPAR-γ agonist e.g. rosiglitazone, would be predicted to be antagonistic to pinometostat activity. Results of experiments investigating the effects of combination of rosiglitazone and pinometostat in MOLM-13 cell lines demonstrated that the combination was significantly less effective at inhibiting cell proliferation than pinometostat alone (IC50 shift of 2 fold (n = 2)). MLL-r fusion partner (e.g. t(11:19)) may influence clinical response to pinometostat. In addition RNASeq based characterization of patient samples and cell lines revealed candidate pathways that may cooperate with or antagonize pinometostat activity that warrant further investigation. Citation Format: Scott Daigle, Alice McDonald, Ty M. Thomson, David A. Drubin, Michael Maria, A. Carson, Brad Patay, Jeff Keats, Christine Klaus, Alejandra Raimondi, G. Garcia-Manero, D. A. Rizzieri, Raoul Tibes, Jesus Berdeja, Eytan M. Stein, Blythe Thomson, Stephen J. Blakemore. Identification of biomarkers and pathways associated with response to the DOT1L inhibitor Pinometostat (EPZ-5676) in MLL-r leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C12.

Exploring drug delivery for the DOT1L inhibitor pinometostat (EPZ-5676): Subcutaneous administration as an alternative to continuous IV infusion, in the pursuit of an epigenetic target

Protein methyltransferases are emerging as promising drug targets for therapeutic intervention in human cancers. Pinometostat (EPZ-5676) is a small molecule inhibitor of the DOT1L enzyme, a histone methyltransferase that methylates lysine 79 of histone H3. DOT1L activity is dysregulated in the pathophysiology of rearranged mixed lineage leukemia (MLL-r). Pinometostat is currently in Phase 1 clinical trials in relapsed refractory acute leukemia patients and is administered as a continuous IV infusion (CIV). The studies herein investigated alternatives to CIV administration of pinometostat to improve patient convenience. Various sustained release technologies were considered, and based on the required dose size as well as practical considerations, subcutaneous (SC) bolus administration of a solution formulation was selected for further evaluation in preclinical studies. SC administration offered improved exposure and complete bioavailability of pinometostat relative to CIV and oral administration. These findings warranted further evaluation in rat xenograft models of MLL-r leukemia. SC dosing in xenograft models demonstrated inhibition of MLL-r tumor growth and inhibition of pharmacodynamic markers of DOT1L activity. However, a dosing frequency of thrice daily (t.i.d) was required in these studies to elicit optimal inhibition of DOT1L target genes and tumor growth inhibition. Development of an extended release formulation may prove useful in the further optimization of the SC delivery of pinometostat, moving towards a more convenient dosing paradigm for patients.

Pediatric Dose Determinations for the Phase I Study of the DOT1L Inhibitor, EPZ-5676, in MLL-r Acute Leukemia: Leveraging Early Clinical Data in Adults through Physiologically-Based Pharmacokinetic Modeling

Introduction: Relapsed/refractory (R/R) MLL-r acute leukemia in children has a poor prognosis, with a reported 5 year overall survival for children less than one year of age with recurrent MLL-r leukemia being 4-20%. The MLL gene, on chromosome 11q23, codes for a histone methyltransferase (HMT) that is responsible for methylation of H3K4, a modification associated with active transcription. Translocations of MLL result in the loss of the SET or catalytic domain of the protein with the most common translocation partners, AF4, AF9, and ENL, recruiting another HMT, DOT1L. The aberrant recruitment of DOT1L to MLL fusion target genes results in ectopic H3K79 methylation and increased expression of genes including HOXA9 and MEIS1, which are involved in leukemogenesis of MLL-rearranged leukemias. EPZ-5676 is a small molecule inhibitor of DOT1L with sub-nanomolar affinity and >37,000 fold selectively against other HMTs. Preclinically, EPZ-5676 selectively inhibits intracellular histone H3K79 methylation, downstream target gene expression and demonstrated complete tumor regressions in an MLL rearranged leukemia xenograft model. The first in human phase I open label study in adult patients with R/R leukemia is ongoing (CT.gov: NCT01684150). Due to the unmet need of MLL-r leukemia in children, we leveraged early clinical data in adult patients to successfully guide dose selection and trial design for a companion pediatric study.Methods: Pediatric starting dose selection was informed using a physiologically-based PK (PBPK) modeling and simulation approach (Simcyp, UK), such that the maturation and developmental aspects of various physiological and biochemical processes important in drug disposition were accounted for e.g. tissue blood flows, organ size, and drug metabolizing enzyme expression. In addition to pharmacokinetic data from adult patients across the dose range of 12 – 90 mg/m2/day administered as a continuous IV infusion (CIV), various in vitro data including physicochemical properties, plasma protein binding, blood partitioning, in vitro metabolic stability and P450 phenotyping were incorporated into the model.Results: Dose proportional pharmacokinetics was observed in adult patients, with rapid attainment of steady-state plasma concentrations on Day 1 of CIV (ASH abstract, 2014). A successful model fit of the adult clinical exposures (n = 16) was achieved that adequately described the time-concentration profiles and PK parameters in terms of population mean and variability. The median clearance observed in adults was 6.00 L/h, and was well predicted using a permeability-limited liver model. System variables related to the pediatric population (organ size, blood flows, enzyme expression) were appropriately adjusted and simulations were performed at a fixed dose across various age groups from 0-18 years. The median clearance predicted in pediatric patients was 0.54, 0.80, 1.09, 1.90 and 4.21 L/h in 1-3 months, 3-6 months, 6 months-2 years, 2-6 years and 6-18 years, respectively. Based on simulations of the predicted steady-state systemic exposure of EPZ-5676 using these pediatric models, dose adjustments of 55%, 65%, 73%, 83% and 100% of the adult dose would be needed to achieve equivalent exposures in the respective age bands. For practical and safety purposes, this was further simplified in a conservative manner to derive a pediatric starting dose of 80% and 50% of the highest adult dose (90 mg/m2/day) in > 12 month olds and < 12 month olds respectively.Conclusions: This study demonstrates that prospective application of modeling and simulation tools, such as PBPK, can support clinical trial design in rare populations early in clinical development, as recently highlighted in FDA’s “Strategic Plan for Accelerating the Development of Therapies for Pediatric Rare Diseases.” These modeling results provided dosing recommendations for the ongoing trial of EPZ-5676 in pediatric MLL-r R/R leukemia which is currently open and enrolling children aged 3 months to 18 years. (CT.gov: NCT02141828). DisclosuresWaters:Epizyme, Inc: Employment, Equity Ownership. Thomson:Epizyme, Inc: Employment. Gardner:Simcyp: Employment. Johnson:Simcyp: Employment. Olhava:Epizyme: Employment. Pollock:Epizyme: Employment, Equity Ownership. Legler:Epizyme, Inc: Employment. Copeland:Epizyme, Inc: Employment, Equity Ownership; Mersana: Membership on an entity’s Board of Directors or advisory committees; New Enterprise Associates: Ad hoc consultant, Ad hoc consultant Other. Hedrick:Epizyme, Inc: Employment; Pharmacyclics, Inc: Equity Ownership.

A distinct set of long non-coding RNAs in childhood MLL-rearranged acute lymphoblastic leukemia: biology and epigenetic target

Long non-coding RNAs (lncRNAs) have been recently found to be pervasively transcribed in human genome and link to diverse human diseases. However, the expression patterns and regulatory roles of lncRNAs in hematopoietic malignancies have not been reported. Here, we carried out a genome-wide lncRNA expression study in MLL-rearranged acute lymphoblastic leukemia (MLL-r ALL) and established lncRNA/messenger RNA coexpression networks to gain insight into the biological roles of these dysregulated lncRNAs. We detected a number of lncRNAs that were differentially expressed in MLL-r ALL samples compared with MLL-r wild-type and identified unique lncRNA expression patterns between MLL-r subtypes with different translocations as well as between infant MLL-r ALL with other MLL-r ALL patients, suggesting that they might be served as novel biomarkers for the disease. Importantly, several lncRNAs that correspond with membrane protein genes, including a lysosome-associated membrane protein, were identified. No such link between the membrane proteins and MLL-r leukemia has been reported previously. Impressively, the functional analysis showed that several lncRNAs corresponded to the expression of MLL-fusion protein target genes, including HOXA9, MEIS1, etc., while some other associated with histone-related functions or membrane proteins. Further experiments characterize the effect of some lncRNAs on MLL-r leukemia apoptosis and proliferation as the function of the coexpressed HOXA gene cluster. Finally, a set of lncRNAs epigenetically regulated by H3K79 methylation were also discovered. These findings may provide novel insights into the mechanisms of lncRNAs involved in the initiation of MLL-r leukemia. This is the first study linking lncRNAs to leukemogenesis.