Preliminary Report of the Phase 1 Study of the DOT1L Inhibitor, Pinometostat, EPZ-5676, in Children with Relapsed or Refractory MLL-r Acute Leukemia: Safety, Exposure and Target Inhibition
Authors: Shukla Neerav , O'Brien Maureen M. , Silverman Lewis B. , Pauly Melinda , Wetmore Cynthia , Loh Mignon L. , Whitlock James A. , Brown Patrick , Thomson Blythe , Blakemore Stephen J. , Daigle Scott , Pimentel Patricia , Waters Nigel J. , Krivstov Andrei V , Koche Richard , Armstrong Scott A. , Ho Peter T , Gore Lia 
Published: October 2019

Abstract
Introduction: MLL-rearranged (MLL-r) acute leukemia in children is characterized by young age at presentation and a poor overall prognosis despite multi-agent chemotherapy. Aberrant fusion proteins involving the MLL histone methyltransferase (HMT) recruit another HMT, DOT1L, to a multi-protein complex leading to aberrant methylation of histone H3 lysine 79 (H3K79) at MLL target genes. This results in enhanced expression of critical genes for hematopoietic differentiation, including HOXA9 and MEIS1, and has been established as a key mechanism for leukemogenesis in MLL-r leukemias (Krivstov, 2007). Pinometostat is a small molecule inhibitor of DOT1L with sub-nanomolar affinity and >37,000 fold selectivity against non-MLL HMTs. Treatment of MLL-rearranged cells and xenograft models with pinometostat led to reduced histone 3, lysine 79 (H3K79) methylation, decreased MLL target gene expression and selective leukemia cell kill (Daigle, 2013). We report the preliminary safety, pharmacokinetics (PK), pharmacodynamics (PD) and target inhibition in leukemia cells of pinometostat in a phase 1 trial in children with relapsed/refractory (R/R) MLL-r acute leukemia.
Methods: An open label dose escalation study of pinometostat was performed in patients (pts) aged 3 months to 18 years (yr) with R/R MLL-r leukemia (NCT02141828). Pinometostat was administered via continuous intravenous infusion until disease progression or unacceptable toxicity. Pts were assigned to one of two aged-based dose escalation schemas developed from simulations of pediatric exposures using a previously reported physiologically-based PK (PBPK) model (Waters, 2014). All patients underwent serial collection of PK and peripheral blood mononuclear cells (PBMC) for PD. Leukemic blasts were isolated from PBMCs using flow cytometry and quantified for di-methylation of H3K79 (H3K79-me2) by ChIP-Seq.
Results: As of 28-June-2015, 11 pts have enrolled in the dose escalation of the study consisting of 10 pts (6 pts at 70 mg/m2/day, 4 pts at 90 mg/m2/day) in the older age cohort (1 to 18 yr) and 1 pt (45 mg/m2/day) in the younger age cohort (<1 yr).
Table 1. Patient Characteristics n (%) Median age, yrs (range) 4 (0.33 - 15) Sex (M / F) 6/5 Diagnosis AML 5 (45) ALL 4 (36) MLL 2 (18) # of prior therapeutic regimens 1 - 2 4 (36) 3 - 7 6 (54) 8 - 10 0 >10 1 (9) Prior allogeneic hematopoietic cell transplant 6 (54)
Adverse events (AEs) reported in >3 pts regardless of attribution were: anemia, febrile neutropenia, abdominal pain, diarrhea, nausea, vomiting, pain, grade 1 prolonged QTcF, lymphopenia, thrombocytopenia, leukopenia, hypocalcemia, hypokalemia, hypophosphatemia, pleural effusion, respiratory failure, dry skin, rash and hypertension. Grade ≥3 related non-hematologic toxicities include: apnea (n=1 and the only protocol defined dose limiting toxicity), organizing pneumonia (n=1), anorexia (n=1), and febrile neutropenia (n=1). The median duration of treatment was 26 days (range 7- 53 days). Updated response data will be provided.
Steady-state plasma concentrations (Css) of pinometostat in children >1 yr at 70 and 90 mg/m2 doses were comparable to that observed in adult patients at equivalent doses and were in the range of 800 - 1600 ng/mL, corroborating earlier PBPK modeling results (projected Css range of 1000-1600 ng/mL at 90 mg/m2/d in ≥1 yr). Steady-state CSF concentrations of pinometostat were low (<2 ng/mL). H3K79-me2 ChIP-Seq demonstrated pinometostat induced reductions in methylation at MLL -r target genes HOXA9 and MEIS1 (range of inhibition = 44-72%)in 2 of 2 pts analyzed to date. Inhibition of H3K79-me2 in leukemic blasts is consistent with DOT1L suppression and additional PK/PD relationships are under investigation.
Conclusions: Pinometostat in children with R/R leukemia has an acceptable safety profile. Exposures of pinometostat in children were consistent with PBPK modeling based on adult exposure data, suggesting that dose/exposure relationships are similar between adults and children > 1 yr. Analysis of H3K79-me2 ChIP-Seq data demonstrated PD reductions in methylation of MLL-r target genes expected from DOT1L inhibition. Enrollment and dose escalation continue.

Disclosures
O'Brien: Seattle Genetics, Inc.: Research Funding. Pauly:Seattle Genetics, Inc.: Research Funding. Whitlock:Amgen: Honoraria. Thomson:Epizyme, Inc: Employment. Blakemore:Epizyme: Employment. Daigle:Epizyme, Inc: Employment. Pimentel:Epizyme, Inc: Employment. Waters:Epizyme, Inc: Employment. Armstrong:Epizyme, Inc: Consultancy. Ho:Epizyme, Inc: Employment.