Bromodomain inhibitor OTX015 in patients with acute leukaemia: a dose-escalation, phase 1 study
Céline Berthon, Emmanuel Raffoux, Xavier Thomas, Norbert Vey, Carlos Gomez-Roca, Karen Yee, David Christopher Taussig, Keyvan Rezai, Christophe Roumier, Patrice Herait, Carmen Kahatt, Bruno Quesnel, Mauricette Michallet, Christian Recher, François Lokiec, Claude Preudhomme, Hervé Dombret
Summary
Background Bromodomain and extraterminal (BET) proteins are chromatin readers that preferentially affect the transcription of genes with super-enhancers, including oncogenes. BET proteins bind acetylated histone tails via their bromodomain, bringing the elongation complex to the promoter region. OTX015 (MK-8628) specifically binds to BRD2, BRD3, and BRD4, preventing BET proteins from binding to the chromatin, thus inhibiting gene transcription. OTX015 inhibits proliferation in many haematological malignancy cell lines and patient cells, in vitro and in vivo. We aimed to establish the recommended dose of OTX015 in patients with haematological malignancies. We report the results of patients with acute leukaemia (leukaemia cohort).
Methods In this dose-escalation, phase 1 study we recruited patients from seven university hospital centres (in France [five], UK [one], and Canada [one]). Adults with acute leukaemia who had failed or had a contraindication to standard therapies were eligible to participate. OTX015 was given orally at increasing doses from 10 mg/day to 160 mg/day (14 of 21 days), using a conventional 3 + 3 design. In this open-label trial, OTX015 was initially administered once a day, with allowance for exploration of other schedules. The primary endpoint was dose-limiting toxicity (DLT), assessed during the first treatment cycle (21 days). The study is ongoing and is registered with ClinicalTrials.gov, NCT01713582.
Findings Between Jan 18, 2013, and Sept 9, 2014, 41 patients, 36 with acute myeloid leukaemia, a median age of 70 years (IQR 60–75) and two lines of previous therapy, were recruited and treated across six dose levels of OTX015. No DLT was recorded until 160 mg/day, when one patient had grade 3 diarrhoea and another had grade 3 fatigue. However, concomitant grade 1–2 non-DLT toxic effects (ie, gastrointestinal, fatigue, or cutaneous) from 120 mg doses hampered patient compliance and 80 mg once a day was judged the recommended dose with a 14 days on, 7 days off schedule. Common toxic effects for all OTX015 doses were fatigue (including grade 3 in three patients) and bilirubin concentration increases (including grade 3–4 in two patients). OTX015 plasma exposure increased proportionally up to 120 mg/day with trough concentrations in the in-vitro active range from 80 mg/day (274 nmol/L). Three patients (receiving 40 mg/day, 80 mg/day, and 160 mg/day) achieved complete remission or complete remission with incomplete recovery of platelets lasting 2–5 months, and two additional patients had partial blast clearance. No predictive biomarkers for response have been identified so far.
Interpretation The once-daily recommended dose for oral, single agent oral OTX015 use in patients with acute leukaemia for further phase 2 studies is 80 mg on a 14 days on, 7 days off schedule.
Funding Oncoethix GmbH, a wholly owned subsidiary of Merck Sharp & Dohme Corp.
Lancet Haematol 2016
Published Online
March 18, 2016 http://dx.doi.org/10.1016/ S2352-3026(15)00247-1
See Online/Comment http://dx.doi.org/10.1016/ S2352-3026(15)00253-7
University Lille, Inserm, CHU Lille, UMR-S 1172—JPArc Centre de Recherche Jean-Pierre AUBERT Neurosciences et Cancer, Lille, France
(C Berthon MD,
C Roumier PharmD, Prof B Quesnel MD,
Prof C Preudhomme PhD);
Hôpital Saint Louis, Assistance
Publique-Hôpitaux de Paris, Paris, France (E Raffoux MD, Prof H Dombret MD); EA-3518,
Institut Universitaire
d’Hématologie, Université Paris Diderot, Paris, France (E Raffoux, Prof H Dombret); Service d’Hématologie, Centre
Hospitalo-Universitaire Lyon-Sud, Pierre
Bénite-Université Claude Bernard Lyon I Faculté de
Médecine Lyon Est, Lyon, France (X Thomas MD,
Prof M Michallet MD); Institut
Paoli Calmettes, Marseille, France (Prof N Vey MD); Université Aix-Marseille, Marseille, France (Prof N Vey); Institut Universitaire du Cancer
Toulouse Oncopole, CHU de
Introduction
Acute myeloid leukaemia is characterised by maturation blockade at an early myeloid progenitor stage, clonal proliferation or accumulation of immature blasts, and bone marrow failure. Although almost half of young (aged <60 years) patients with acute myeloid leukaemia can be cured,1 patients who are refractory or relapsing, or not fit for intensive therapy (mainly elderly patients, aged
>60 years) have few therapeutic options.
Epigenetic deregulation is thought to be an important factor in acute myeloid leukaemia genesis, with many recurrent gene changes affecting transcriptional activity in both acute myeloid leukaemia and myelodysplastic syndromes.2–4 Hypomethylating drugs, including decitabine and azacitidine, were the first epigenetic modulators approved in myeloid malignancies.5–8 Nonetheless, with
little proven activity, other epigenetic drug families are being investigated, including histone deacetylase (HDAC), isocitrate dehydrogenase (IDH), and bromo- domain (BRD) inhibitors.
Bromodomain and extraterminal (BET) proteins are chromatin readers that play a major part in the epigenetic regulation of gene transcription.9 Their bromodomain- bearing moiety binds to acetylated histone tails, allowing the extraterminal moiety to bring the elongation complex of the transcriptional machinery within proximity of the gene promoter region. Histone acetylation is prevalent at super-enhancer regions that control expression of various oncogenes, rendering them sensitive to bromodomain inhibition.10 Bromodomain inhibitors are small molecules that specifically bind bromodomains,11 preventing BET proteins from binding to chromatin and thereby inhibiting
Toulouse, Université de Toulouse III, IUCT-O, Toulouse, France (C Gomez-Roca MD,
Prof C Recher MD); Princess
Margaret Cancer Center,
Toronto, ON, Canada
(K Yee MD); Royal Marsden Hospital, Sutton, Surrey, UK (D C Taussig MD); Institute of Cancer Research, Sutton, UK (D C Taussig); Département de Radio-Pharmacologie, Institut
Curie, Hôpital René Huguenin,
Saint-Cloud, France
(K Rezai PhD, F Lokiec ScD); Oncoethix SA (now Oncoethix
GmbH), Lucerne, Switzerland (P Herait MD); and Oncology Therapeutic Development, Clichy, France (C Kahatt MD)
Research in context
Evidence before this study search terms “leukaemia”, “bromodomain inhibitor”, and Intensive cytarabine-based and anthracycline-based induction “patient/clinical”.
therapy, followed by intensification with or without allogeneic Added value of the study
stem-cell transplantation, allows about 50% of younger To our knowledge, this is the first clinical evidence of the novel (aged <60 years) patients with acute myeloid leukaemia to be first-in-class bromodomain inhibitor OTX015 (MK-8628) cured. Nonetheless, the outcome for many patients remains administered to patients with advanced acute leukaemia. poor, either because they are excluded from intensive standard OTX015 was safely administered at 80 mg once a day with a
induction and consolidation therapies (older patients, aged discontinuous schedule. Evidence of activity was noted in
≥60 years), or because their disease is refractory or relapses after several patients with acute myeloid leukaemia treated with such therapies. Of novel therapeutic approaches, epigenetic OT0X15 doses giving plasma exposures equivalent to those manipulation has been explored with hypomethylating drugs resulting in activity in in-vitro studies.
and in the past 10 years by inhibition of bromodomains,
histone deacetylase, and isocitrate dehydrogenase (IDH). Implications of all the available evidence
Bromodomain inhibition has shown promising results in In view of reported activity in an elderly population with acute preclinical studies in acute myeloid leukaemia cell lines, myeloid leukaemia along with a manageable safety profile, including specific acute myeloid leukaemia subsets such as future development is merited for this bromodomain inhibitor those with NPM1 or IDH2 gene mutations, MLL rearrangement, in combination with other compounds active in this
or EVI1 overexpression. So far no clinical evaluations of population. Extended genetic molecular biology analyses will
bromodomain inhibition have been published. We searched help guide the selection of populations most likely to benefit PubMed for articles published before July 1, 2015, using the from this class of compound.
Correspondence to: Prof Hervé Dombret, Institut Universitaire d’Hématologie, Hôpital Saint-Louis, 75010 Paris,
France
[email protected]
See Online for appendix
gene transcription. These inhibitors have raised much interest as a promising, novel therapeutic approach in acute myeloid leukaemia12–17 and other cancers. In acute myeloid leukaemia, several potential biomarkers for bromodomain inhibition efficacy have been reported. The NPM1 gene mutation, present in 25–30% of adults with acute myeloid leukaemia, favours a BRD4-dependent core transcriptional programme that renders cells particularly sensitive to bromodomain inhibition.16 Less frequent abnormalities (eg, IDH2 mutation,15 EVI1 overexpression,17 or MLL rearrangement14) have been reported as potential predictors, based on preclinical models.
OTX015 (MK-8628 [Oncoethix GmbH, a wholly owned subsidiary of Merck Sharp & Dohme Corp], Lucerne, Switzerland) is a 528 Da molecule that binds exclusively to BRD2, BRD3, and BRD4 with an EC50 of 10–19 nmol/L, inhibiting their binding to acetylated histone H4 with an IC50 of 92–112 nmol/L.18 It exhibits anti-proliferative effects against a large panel of haematological malignancy cell lines with a GI50 of 40–500 nmol/L. Ten of 17 leukaemia cell lines tested were deemed sensitive with an IC50 less than 500 nmol/L after 72 h exposure.19 Furthermore, primary bone marrow cells from eight of 14 patients with acute myeloid leukaemia were sensitive to OTX015 at similar concentrations, and apoptosis was also reported. Although growth inhibition has been noted with various in-vivo tumour models,20 animal models of acute leukaemia have yet to be explored.
Haematological, gastrointestinal, and hepatic toxic effects were limiting at the highest OTX015 doses tested in toxicology studies, with no cumulativity of effects noted up to 12 months. No specific target organs were identified in preclinical, safety pharmacology studies. The primary
aim of this study was to establish the recommended dose of OTX015 in two independent and parallel cohorts, one in patients with acute leukaemia and one in those with non-leukaemic haematological malignancies. We report results for the acute leukaemia cohort.
Methods
Study design and patients
We did a dose-escalation, phase 1 study in seven university hospital centres (in France [five], UK [one], and Canada [one]; appendix p 1). The study was designed with two independent and parallel cohorts, one in patients with acute leukaemia and the other in patients with non-leukaemic haematological malignancies. We report results for the acute leukaemia cohort. Data for the non-leukaemia cohort are published elsewhere.21
To be eligible to participate in this trial, patients had to be aged 18 years or older and have relapsed or refractory acute leukaemia (patients with promyelocytic leukaemia were not eligible). Patients aged younger than 60 years had to have failed at least two standard regimens, whereas older patients had to have failed at least one regimen and have relapsed within 1 year after previous first-line therapy, or have a contraindication for standard therapies to be eligible. Previous allogeneic stem-cell transplantation was allowed, provided relapse had occurred more than 90 days later. Symptoms or treatment of graft-versus-host disease were not permitted at study entry. Patients were required to have an Eastern Cooperative Oncology Group performance status of 0–2, normal bilirubin concentrations, amino- transferases at three times or less the upper limit of normal, creatinine clearance of 30 mL/min or more,
and have a life expectancy of 3 months or longer. For full inclusion and exclusion criteria see protocol (appendix). Patients were recruited from hospitals by their treating physician. All patients provided written informed consent. Regulatory approval was obtained from all national authorities, and all local institutional review boards approved the protocol (appendix) as appropriate. The study was conducted in accordance with the Declaration of Helsinki.
Procedures
We started with a once a day schedule and allowed for exploration of other dosing schedules according to the decision of a safety monitoring committee. Eligible patients received OTX015 delivered orally as 10 mg or 20 mg gelatine capsules, under fasted conditions, once a day for 14 days consecutive, followed by a 1-week rest without OTX015 (21-day cycles), at a starting dose of 10 mg once a day. A conventional 3 + 3 dose-escalation schedule was used with three patients initially per dose level. Doses were escalated after three patients had received at least one cycle as planned without any dose-limiting toxicities (DLTs) reported. Doses were doubled until DLTs were reported at which point three additional patients were treated at this dose. If no more than one of the six patients had a DLT, dose escalation continued to be increased as based on the more conservative Fibonacci-like model. In this open-label study, the highest dose at which no more than one of six patients had a DLT was deemed the recommended dose for OTX015. On the basis of preclinical models, the possibility that continuous exposure at active concentrations was necessary for optimal activity was assessed after 80 mg once a day, with a twice a day schedule (40 mg given two times a day).
DLT was defined as any one of the following occurring during the first cycle (21 days): as defined by National Cancer Institute Common Toxicity Criteria, a grade 3 or
4 non-haematological-related event, despite optimum supportive care; grade 3 or 4 asymptomatic non- haematological laboratory abnormality lasting for more than 7 days; blast-free aplasia confirmed as lasting for 42 days or longer, or a grade 2 event leading to dose interruption or adaptation. If such toxic events were reported at any time during treatment, OTX015 was interrupted until recovery to grade 1 or lower (or baseline value) and treatment resumed at one dose level lower. A safety monitoring committee (consisting of all principal investigators, the sponsor’s medical and safety officers, and an independent expert) made all decisions regarding patient safety, DLT qualification, dose escalation, and recommended dose definition. Treatment was continued until progression, intolerable toxic effects, treatment interruption for more than 2 weeks due to toxic effects, DLT recurrence despite dose reduction, or patient withdrawal of consent.
Standard haematology and biochemistry work-ups were done once every week during the first cycle, then
once every 3 weeks in the absence of toxic effects, and 12-lead electrocardiograms were completed before first dose and about the time to peak concentration (Tmax) on cycle 1 day 1. Bone marrow aspirations were taken at baseline, and at days 8, 22, and 43, and thereafter if requested by the physician. Patient response was assessed according to European LeukemiaNet.22 Partial blast clearance was defined as a greater than 50% reduction in bone marrow blast percentage without persistent circulating blasts.
Mutational analyses were completed in DNA from bone marrow samples taken at baseline to assess a panel of 42 genes commonly mutated in acute myeloid leukaemia and myelodysplastic syndromes (appendix p 2), using a MiSeq platform (Illumina, San Diego, CA, USA). The library was prepared with the Haloplex Target Enrichment System (Agilent Technologies, Santa Clara, CA, USA).
Plasma OTX015 concentrations were measured in blood samples (3 mL) collected into K2-EDTA (edetic acid) tubes before the first OTX015 administration, and then at 1 h, 4 h, 6 h, 8 h, 12 h, 16 h, and 24 h after drug intake. Samples were immediately centrifuged and stored at –20°C. Samples for residual plasma concentration were collected just before dosing on days 8, 15, and 22 after treatment initiation. Intracellular OTX015 concentrations were measured from bone marrow and blood monocytes, which were collected on day 8 immediately before dosing. Bone marrow and blood were collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), stored at room temperature (20–25°C), and mononuclear cells were separated on a Ficoll gradient. OTX015 concentrations were measured by a validated method using ultra performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS).23 Data analyses were completed with Monolix software (version 4.3).
Outcomes
The primary endpoint was to establish the recommended dose of OTX015 for further phase 2 studies, in patients with acute leukaemia and in patients with other haematological malignancies. Secondary endpoints were the safety profile and pharmacokinetics of OTX015, evidence of its clinical anti-tumour activity, and potential predictive biomarkers for efficacy.
Statistical analysis
Patients receiving at least 85% of the cumulative intended dose over the first 21 days were evaluable for DLTs. All treated patients were evaluated for safety. Patients were evaluable for efficacy according to the European LeukemiaNet.22 Descriptive analyses were completed with a cutoff of Feb 14, 2015, using SAS software (version 9.4). This study is ongoing and is registered with ClinicalTrials.gov, number NCT01713582.
Role of the funding source
The funder of the study designed the study in collaboration with the investigators and wrote the manuscript with the corresponding author. Data collection, data analysis, and data interpretation were completed by the investigating
0 13 (32%)
1 24 (58%)
2 4 (10%)
(n=41) (n=37)* DLT
1 10 mg QD (14 of 21 days) 3 3 0
2 20 mg QD (14 of 21 days) 3 3 0
3 40 mg QD (14 of 21 days) 4 4 0
4a 80 mg QD (14 of 21 days) 4 4 0
research teams, the study contract research organisation, and the funder. Investigators had access to all the data in the study and had final responsibility for the decision to submit for publication. The manuscript was approved by all authors.
Results
Patients were recruited between Jan 18, 2013, and Sept 9, 2014. 40 patients with acute leukaemia and one patient with high-risk myelodysplastic syndrome were enrolled and treated with OTX015 at six doses. The population was mainly elderly and most patients had acute myeloid leukaemia, more than a third of whom were secondary to pre-leukaemic conditions or therapy-related conditions (table 1).
In the dose escalation, doses of OTX015 were doubled without DLTs reported at the first four doses (10 mg, 20 mg, 40 mg, 80 mg once a day; dose levels 1–4a; table 2). To test biological activity of this inhibitor with continuous exposure at high concentrations, a twice a day schedule was explored at dose level 4b (40 mg twice a day). Although no DLTs were reported, assessment of the parallel cohort of patients with non-leukaemic haematological diseases showed an excess of thrombo- cytopenia without an obvious effect on activity for this twice a day schedule,21 and increased frequency of non-haematological toxic effects (eg, gastrointestinal). Additionally, two of eight patients without severe thrombocytopenia at baseline had aggravation of thrombocytopenia during twice a day treatment, needing treatment interruption and dose adaptation; treating physicians decided against continuing OTX015 treatment with 40 mg twice a day for both cohorts. Furthermore, given that clinical responses were noted in patients with leukaemia who received the once a day schedule (and not with the twice a day schedule), dose escalation was resumed with a once a day dosing at 120 mg. No DLTs were reported in the first three patients treated. At 160 mg OTX015 once a day, two of five evaluable patients had DLTs, one had grade 3 fatigue and one had grade 3 diarrhoea (table 3). 160 mg at once a day was therefore deemed not tolerable and additional patients were enrolled at 120 mg once a day to show tolerance and optimise the schedule with four patients treated with the original intermittent once a day schedule (14 of 21 days) and six patients with a continuous schedule (21 of 21 days). Although DLT was not reported in the 13 patients treated at 120 mg (irrespective of schedule), the safety monitoring committee considered that the cumulative prevalence of several gastrointestinal events, cutaneous events, and fatigue was poorly tolerated in this elderly, heavily pretreated population with frequent comorbidities, hampering patient compliance (table 3). Furthermore, active plasma trough concentrations achieved at 80 mg/day along with no clear dose-effect association with clinical response led to the selection of 80 mg once
40 mg (QD, 14 of 80 mg (QD, 14 of 40 mg (BID, 14 of 120 mg (QD, 14 of 120 mg (QD, 21 of 160 mg (QD, 14 of
21 days; n=4) 21 days; n=4) 21 days; n=8) 21 days; n=7) 21 days; n=6) 21 days; n=6)
Grade 1–2 Grade 3–4 Grade 1–2 Grade 3–4 Grade 1–2 Grade 3–4 Grade 1–2 Grade 3–4 Grade 1–2 Grade 3–4 Grade 1–2 Grade 3–4
Diarrhoea 1 0 0 0 1 0 3 0 4 0 4 1
Nausea 1 0 1 1 2 0 1 0 2 0 1 0
Anorexia 0 0 0 0 2 0 1 0 2 0 2 0
Vomiting 0 0 0 1 0 0 1 0 1 0 0 0
Skin disorders 0 0 0 0 0 0 4 0 2 0 2 0
Fatigue 1 1 0 0 1 0 4 0 1 0 1 2
Factor VII decrease 1 0 0 0 2 0 1 0 1 0 0 0
Direct bilirubin increase 0 0 0 0 0 0 0 1 0 2* 0 0
Aminotransferase elevation 0 0 0 0 0 1* 0 0 0 0 0 0
QD=once a day. BID=twice a day. *Grade 4 in one patient. No grade 5 adverse events reported. Only grade 1–2 events were reported in patients treated with 10 mg and 20 mg.
Table 3: Related non-haematological adverse events in more than 10% of patients (any grade) and all grade 3–5 related events
a day given for 14 days and 7 days off to be the
recommended dose (table 4).
Patients received a median of two 21-day cycles (IQR 1–4) of OTX015. Three patients (treated at 40 mg twice a day and 160 mg once a day) had dose reductions Plasma Blood and bone marrow
Evaluable C ; day 1, C pre-dose; AUC day 1, Evaluable Pre-dose cycle 1 day 8, cycle 1 cycle 1 (μg × h per
due to grade 2–3 non-haematological toxic effects. Table 3 (nmol/L)
shows the main adverse events deemed related to 10 mg (QD) 3 258 (154) 29·0 (18·4) 1218 (161) 2 1·35
OTX015. Few adverse events with OTX015 treatment 20 mg (QD) 3 557 (230) 102 (1617) 3171 (876) 3 1·05
(all of which were grade 1 or 2) were reported with the 40 mg (QD) 4 1522 (854) NA* 5792 (1347) 1 0·20
first two dose levels (once a day 10 mg or 20 mg). 80 mg (QD) 5† 2230 (931) 274 (142)‡ 12 890 (4353) 3 2·27
The most common adverse events reported were 40 mg (BID) 8 1028 (348) NA* 14 410 (4743) NA NA
gastrointestinal (diarrhoea: 14 [34%] patients, and nausea: 120 mg (QD) 12 3756 (1646) 585 (364)§ 18 730 (10 490) 7 2·70
nine [22%] patients), fatigue (11 [27%] patients), and 160 mg (QD) 5 2627 (1162) 437 (386)¶ 14 090 (3761) 3 3·67
cutaneous (eight [20%] patients); frequency and severity of these toxicities increased with doses greater than
80 mg once a day, including at 40 mg twice a day. Asymptomatic laboratory abnormalities included grade 1–2 factor VII decrease (lowest values of 20–30% with little effect on international normalised ratio) noted in five patients without associated coagulation factor deficiency. Positive de-challenge and re-challenge in one patient showed the association with study treatment; however, treatment discontinuation was not necessary. Three patients had isolated (without other liver test dysfunctions) direct bilirubin concentration increases (all grade 3–4 adverse events and generally associated with neutropenic sepsis), with a positive de-challenge or re-challenge in one patient suggesting that this might be associated with use of OTX015. Another patient had recurrent transient grade 3 elevation of aminotransferases, although this patient had a similar result when previously treated with chemotherapy for breast cancer. Grade 3 or 4 toxic effects were infrequent (reported in ten [30%] of 33 patients), all of which were reversed within 10 days of stopping the drug. Peripheral blood cytopenia was not considered for the safety assessment, nevertheless in several patients without severe thrombocytopenia at baseline, aggravation of thrombocytopenia occurred during study treatment. No substantial changes in QTc interval were recorded. There were no treatment-related
deaths and no patients discontinued treatment because of related adverse events.
Table 4 shows the pharmacokinetic characteristics for all patients evaluable for these parameters. Plasma exposure increased proportionally with dose from 10 mg to 120 mg given once a day. Terminal half-life was 5·79 h (SD 1·12).24 No accumulation trend was noted until day 15. Intracellular OTX015 concentrations were measured on day 8 pre-dose in 19 (46%) of 41 patients and showed low concentrations, in the nanomolar range. Although intracellular concentrations increased with dose, they remained in the single-digit nanomolar range, and the concentration increase was much lower in cells (three to four times from 10 mg to 160 mg of OTX015) than in plasma (30–40 times from 10 mg to 160 mg).
Drug activity was recorded at a range of dose levels. Two patients had complete remission—one had acute myeloid leukaemia (40 mg once a day) and one had refractory anaemia with excess of blasts (160 mg once a day). A third patient with acute myeloid leukaemia had a
Absolute change in neutrophils vs baseline (109/L)
Figure 1: Waterfall plots of maximum absolute variations during OTX015 treatment versus baseline per patient with acute leukaemia
Percentage bone marrow blasts (n=37 patients; four patients did not have post-baseline blasts) and (B) blood neutrophil counts (n=34 patients; seven patients were missing baseline or first follow-up values). CR=complete remission. CRi=complete remission with incomplete recovery of platelets. DL=dose level. QD=once a day. BID=twice a day.
Absolute variation in bone marrow blasts vs baseline (%)
complete remission with incomplete recovery of platelets (80 mg once a day; appendix p 3). Two other patients with acute myeloid leukaemia secondary to polycythemia vera (treated with 10 mg once a day) and myelodysplastic syndrome (80 mg once a day) had partial blast clearance. The patient treated with 80 mg of OTX015 once a day also had a transient increase of neutrophil counts greater than 1·0 × 10⁹ cells per L. All three patients with responses relapsed on therapy; the two patients with complete remissions lasting 5 months and 3 months, and the third patient who had complete remission with
incomplete recovery of platelets lasting 2 months. The patient with refractory anaemia with excess of blasts treated at 160 mg once a day had long-lasting treatment interruptions that could potentially have contributed to relapse. Figure 1A shows maximum variation in bone marrow blast percentages per patient during treatment and figure 1B shows maximum variation in absolute neutrophil counts. 13 patients had a maximum absolute neutrophil count increase of more than 2 × 10⁹ cells per L during treatment compared with baseline, including non-responding patients.
Figure 2: 42-gene panel mutational profile in responders versus non-responders
Five patients had evidence of clinical activity (A; three had complete remissions and two had partial blasts clearance), which did not show a clearly different mutational profile compared with the other 28 patients with no evidence of clinical activity (B). Orange boxes indicate mutated genes. Blue boxes indicate the absence of mutation.
G-G 06626
H-A 03323
D-B 962
M-A 01120
G-M 03302
D-P 03350
O-A 971
A-M 101059
G-O 951
A-C 03306
H-M 06681
M-R 06631
D-M 01
G-C 03354
G-M 01155
F-F 01117
LJC 03316
L-J 03308
M-M 01104
J-B 01174
G-B 01
A-P 06640
M-G 101029
J-J 101041
M-M-V 01173
JC-B 101077
G-S 967
R-G 03353
WJP 01148
C-J 03343
F-L 03330
J-V 01101
G-S 03372
Genetic analyses did not show correlations between mutations present in the five patients who responded versus those in the 28 non-responders with genetic analyses at study entry (figure 2). Of note, neither the NPM1 mutations (one patient with NPM1 mutation was a responder vs six non-responders with this mutation) or IDH2 mutations (no responders of two mutated patients) correlated with clinical activity of OTX015. One responding patient was FLT3 wild-type, whereas three of the six non-responders had FLT3 internal tandem duplication. No hints of clinical activity were noted in three patients with EVI1 overexpression or the two patients with MLL rearrangement.
Discussion
We showed that OTX015 was safely administered at a dose of 80 mg, once a day on a 14 days on 7 days off schedule, in pretreated patients with acute leukaemia, with linear pharmacokinetics for the doses evaluated up to 120 mg. Above 80 mg once a day, plasma concentrations were maintained above 400 nmol/L, a concentration known to be active in vitro.19 Nonetheless, low OTX015 intracellular concentrations were reported in day 8 bone marrow and peripheral blood monocyte samples, compared with trough plasma concentrations. This finding is coherent with preclinical studies reporting that 500 nmol/L extracellular in-vitro concentrations resulted
in intracellular concentrations about ten to 50 times lower at which anti-proliferative activity was noted.25 Taken together, in-vitro and clinical data show that low intracellular concentrations of OTX015 in the nanomolar range can be active.
Four of the five patients with anti-leukaemic activity were treated at 80 mg once a day or at a lower dose, offering pharmacodynamic evidence (in the absence of known off-target effects) that OTX015 does indeed attain its targets and triggers a biological effect in this dose range. Increasing the dose above this level increased toxic effects without a clear effect on efficacy. The patient with refractory anaemia and an excess of bone marrow blasts who had complete remission (160 mg once a day), had recurrent toxic effects that led to extended treatment interruption periods, potentially jeopardising efficacy and duration of response.
From preclinical data showing a rapid washout effect,20 we anticipated that increased use of high concentrations, equal to or higher than active in-vitro concentrations, would be necessary for optimum clinical activity. A twice a day schedule was thus tested to increase trough concentrations, achieving similar exposure as the equivalent once a day dose. Although a twice a day schedule for continuous exposure is desirable, the excess of thrombocytopenia and various non-haematological toxicities reported in the cohort receiving twice a day doses resulted in the decision by the safety monitoring committee not to pursue treating patients with this schedule at higher doses. No evidence of clinical activity was recorded in the eight patients treated with the twice a day schedule. Additionally, decisions made during the study involving pharmacokinetic data accounted for data from the non-leukaemic cohort, for which some trough concentration data were available.21 A twice a day schedule offers the advantage of erasing peak con- centrations, but no evidence is available to show that any of the toxic effects reported are peak related. In the absence of evidence of a benefit with a twice a day rather than a once a day schedule, the once a day delivery was selected to optimise patient convenience and compliance.
OTX015-related adverse events were manageable, because most were mild to moderate at the recommended dose, whereas severe toxicity rapidly resolved after drugs were stopped. The range of toxic effects (ie, gastro- intestinal, fatigue, and cutaneous) resembles that of HDAC inhibitors,26 another family of epigenetic drugs. Haematological toxic effects, mainly thrombocytopenia, associated with OTX015 are dose-limiting in the non-leukaemic cohort.21 These effects are not easily assessed in patients with acute leukaemia, but the few patients with incomplete bone marrow failure at baseline developed severe thrombocytopenia. Similarly, the patient treated at 40 mg who achieved extended complete remission had grade 1 thrombocytopenia at
day 15 during remission. The patient recovered completely after 1 week off treatment. Several cases of isolated, asymptomatic factor VII decreases were reported in the absence of other vitamin K-dependent factor deficiencies, which was also noted in patients with lymphoma or myeloma.21 In-vitro analyses in human HepaRG hepatocyte-like cells exposed to OTX015 suggest that the underlying mechanism includes inhibition of factor VII gene expression (Francesco Bertoni, Institute of Oncology Research, Bellinzona, Switzerland, personal communication). Although two of the three patients with hyperbilirubinaemia had factor VII decreases, these events are unlikely to be connected, with hyper- bilirubinaemia probably caused by reduced bilirubin transport across the canalicular membrane after MRP/ABCC2 downregulation or by competitive exclusion of bilirubin due to extrusion of OTX015 via the MRP/ABCC2 transporter.27–29
During dose escalation, four patients were treated at 80 mg once a day without a DLT. With 13 patients at
120 mg (all once a day; different administration schedules), all without a DLT, 80 mg once a day was established by the safety monitoring committee as the recommended dose at which additional patients were to be evaluated in an expansion cohort. Taken together, the treatment-limiting thrombocytopenia and recovery (which was incomplete in some cases) with a 1-week break led to the recommendation of an intermittent schedule to ensure an adequate opportunity to recover from any toxic effects. Although a so-called drug holiday could affect drug efficacy, responses were noted with an intermittent schedule, which was deemed necessary not only from a safety perspective but also to allow long- term treatment. Additionally, evaluation of twice a day schedules at doses less than the recommended dose of 80 mg once a day is anticipated to address presumed on- target effects of thrombocytopenia and the importance of continuous sustained exposure (steady state Ctrough), based on the mechanism of action of OTX015.
Preliminary molecular biology analyses did not detect any clinical or genetic predictive factors of clinical activity from the panel of candidates assessed in this acute leukaemia population, including in patients with putative predictive markers based on preclinical modelling data. Given the small sample size, further efforts are needed to elucidate which patients are most likely to respond to bromodomain inhibition, so as to enrich populations in future clinical trials. Although many epigenetic changes are driven by genetic alterations, epigenetic rather than genomic investigations might be more successful. Pharmacodynamic analyses that assess the expression of seven oncogenes (c-MYC, BCL2, CCND1, NF-κB, BRD2, BRD3, and BRD4) in bone marrow cells at baseline and after 1 week of treatment were done (results not shown). mRNA downregulation was not noted for any of the seven genes evaluated in all patients, including those treated at active doses and those with clinical responses.
Because c-MYC mRNA has been shown to be downregulated in vitro in both leukaemic cell lines and in patient bone marrow cells at OTX015 concentrations achieved in the plasma of patients treated at active doses,19 these clinical data were judged difficult to interpret. This discrepancy and the failure to show mRNA downregulation in vivo is not fully understood, but might be connected to the delay between bone marrow collection and nucleic acid extraction. Additionally, low cellularity and a mixture of leukaemic and non-leukaemic cells in bone marrow samples might account for discrepancies between in-vitro and in-vivo testing.
Clinical activity of bromodomain inhibition alone might be insufficient to successfully manage this patient population. All responding patients relapsed and, importantly, several additional patients not formally deemed as responders showed transient decreases in blasts and increases in neutrophil concentrations, suggesting the potential value of combination therapy. In support of this clinical developmental route, additive or synergistic activity of OTX015 has been reported in vitro with several cytotoxic drugs and other epigenetic drugs, such as HDAC inhibitors and hypomethylating agents, against leukaemia cell lines.30 Combination trials are anticipated, favouring hypomethylating agents in myeloid malignancies due to possible cumulative toxicity with HDAC inhibitors.
With five patients with acute leukaemia showing evidence of clinical activity, including three responses, in a fragile, elderly, heavily pretreated population, to our knowledge this study is the first demonstration of a single agent BET-bromodomain inhibitor showing clinical activity in patients with refractory or resistant acute myeloid leukaemia or myelodysplastic syndrome, rendering further development of this new family of agents of great interest in myeloid malignancies. With few responders in an unselected population, the two most reliable pathways for future research for OTX015 use are identification of predictive biomarkers and combination therapy.
Contributors
PH and HD were involved in the study design. CB, ER, XT, NV, CG-R, KY, DCT, CRo, BQ, MM, CRe, and HD did data collection. KR, PH, CK, FL, and CP did the data analysis and data interpretation. PH and HD wrote the manuscript. All authors reviewed and approved the final version of the manuscript.
Declaration of interests
CB, ER, XT, NV, CG-R, KY, DCT, KR, CRo, BQ, MM, CRe, FL, CP, and HD
received institutional funding from Oncoethix SA for this clinical trial. PH is a former stockholder and chief medical officer of Oncoethix SA. CK is an employee of the CRO, managing the study on behalf of Oncoethix SA.
Acknowledgments
François Montestruc (eXYSTAT, Malakoff, France) did statistical analyses and prepared figures, and Sarah MacKenzie (Oncology Therapeutic Development, Clichy, France) did manuscript editing. We thank the patients and their families, hospital teams, the team at Oncology Therapeutic Development, the CRO in charge of the study, and the safety monitoring committee. DCT is supported by funding from the National Institute for Health Research RM/ICR Biomedical Research Centre.
References
Burnett AK. Treatment of acute myeloid leukemia: are we making progress? Hematology Am Soc Hematol Educ Program 2012;
2012: 1–6.
Shih AH, Abdel-Wahab O, Patel JP, Levine RL. The role of mutations in epigenetic regulators in myeloid malignancies. Nat Rev Cancer 2012; 12: 599–612.
Mehdipour P, Santoro F, Minucci S. Epigenetic alterations in acute myeloid leukemias. FEBS J 2015; 282: 1786–800.
Cancer Genome Atlas Research Network. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N Engl J Med 2013; 368: 2059–74.
Fenaux P, Mufti GJ, Hellstrom-Lindberg E, et al, for the International Vidaza High-Risk MDS Survival Study Group. Efficacy of azacitidine compared with that of conventional care regimens in the treatment of higher-risk myelodysplastic syndromes: a randomised, open-label, phase III study. Lancet Oncol 2009; 10: 223–32.
Fenaux P, Mufti GJ, Hellström-Lindberg E, et al. Azacitidine prolongs overall survival compared with conventional care regimens in elderly patients with low bone marrow blast count acute myeloid leukemia.
J Clin Oncol 2010; 28: 562–69.
Kantarjian HM, Thomas XG, Dmoszynska A, et al. Multicenter, randomized, open-label, phase III trial of decitabine versus patient choice, with physician advice, of either supportive care or low-dose cytarabine for the treatment of older patients with newly diagnosed acute myeloid leukemia. J Clin Oncol 2012; 30: 2670–77.
Dombret H, Seymour JF, Butrym A, et al. International phase 3 study of azacitidine vs conventional care regimens in older patients with newly diagnosed AML with >30% blasts. Blood 2015;
126: 291–99.
Filippakopoulos P, Knapp S. Targeting bromodomains: epigenetic readers of lysine acetylation. Nat Rev Drug Discov 2014; 13: 337–56.
Lovén J, Hoke HA, Lin CY, et al. Selective inhibition of tumor oncogenes by disruption of super-enhancers. Cell 2013;
153: 320–34.
Filippakopoulos P, Qi J, Picaud S, et al. Selective inhibition of BET bromodomains. Nature 2010; 468: 1067–73.
Zuber J, Shi J, Wang E, et al. RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia. Nature 2011; 478: 524–28.
Herrmann H, Blatt K, Shi J, et al. Small-molecule inhibition of BRD4 as a new potent approach to eliminate leukemic stem- and progenitor cells in acute myeloid leukemia AML. Oncotarget 2012; 3: 1588–99.
Zhang Y, Chen A, Yan X-M, Huang G. Disordered epigenetic regulation in MLL-related leukemia. Int J Hematol 2012; 96: 428–37.
Chen C, Liu Y, Lu C, et al. Cancer-associated IDH2 mutants drive an acute myeloid leukemia that is susceptible to Brd4 inhibition. Genes Dev 2013; 27: 1974–85.
Dawson MA, Gudgin EJ, Horton SJ, et al. Recurrent mutations, including NPM1c, activate a BRD4-dependent core transcriptional program in acute myeloid leukemia. Leukemia 2014; 28: 311–20.
Gröschel S, Sanders MA, Hoogenboezem R, et al. A single oncogenic enhancer rearrangement causes concomitant EVI1 and GATA2 deregulation in leukemia. Cell 2014; 157: 369–81.
Noel JK, Iwata K, Ooike S, Sugahara K, Nakamura H, Daibata M. Development of the BET bromodomain inhibitor OTX015.
Mol Cancer Ther 2013; 12 (suppl): C244 (abstr).
Coudé M-M, Braun T, Berrou J, et al. BET inhibitor OTX015 targets BRD2 and BRD4 and decreases c-MYC in acute leukemia cells. Oncotarget 2015; 6: 17698–712.
Boi M, Gaudio E, Bonetti P, et al. The BET bromodomain inhibitor OTX015 affects pathogenetic pathways in preclinical B-cell tumor models and synergizes with targeted drugs. Clin Cancer Res 2015; 21: 1628–38.
Amorim S, Stathis A, Gleeson M, et al. Bromodomain inhibitor OTX015 in patients with lymphoma or multiple myeloma: a dose- escalation, open-label, pharmacokinetic, phase 1 study. Lancet Haematol 2016; published online March 18. http://dx.doi. org/10.1016/S2352-3026(16)00021-1.
Döhner H, Estey EH, Amadori S, et al. Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet. Blood 2010; 115: 453–74.
Odore E, Lokiec F, Weill S, et al. Development and validation of an UPLC-MS/MS method for quantitative analysis of OTX015 in human plasma samples. Anal Methods 2014; 6: 9108–15.
Odore E, Lokiec F, Cvitkovic E, et al. Phase I population pharmacokinetic assessment of the oral bromodomain inhibitor OTX015 in patients with haematologic malignancies.
Clin Pharmacokinet 2015; published online Sept 4. DOI:10.1007/ s40262-015-0327-6.
Odore E, Astorgues-Xerri L, Bekradda M, et al. Cellular pharmacokinetics and molecular pharmacodynamics studies of the BRD-BET inhibitor OTX015 in sensitive and resistant leukemic cell lines. Eur J Cancer 2014; 50 (suppl 6): 189 (abstr 587).
Mottamal M, Zheng S, Huang TL, Wang G. Histone deacetylase inhibitors in clinical studies as templates for new anticancer agents. Molecules 2015; 20: 3898–941.
Hussong M, Börno ST, Kerick M, et al. The bromodomain protein BRD4 regulates the KEAP1/NRF2-dependent oxidative stress response. Cell Death Dis 2014; 5: e1195.
Kim H, Kim S-N, Park Y-S, et al. HDAC inhibitors downregulate MRP2 expression in multidrug resistant cancer cells: implication for chemosensitization. Int J Oncol 2011; 38: 807–12.
Vasilyeva A, Durmus S, Li L, et al. Hepatocellular shuttling and recirculation of sorafenib-glucuronide is dependent on Abcc2, Abcc3, and Oatp1a/1b. Cancer Res 2015; 75: 2729–36.
Astorgues-Xerri L, Canet-Jourdan C, Bekradda M, et al. OTX015, a BET-bromodomain (BET-BRD) inhibitor, potentiates the in vitro effects of chemotherapy drugs and targeted agents in human leukemic cell lines. Eur J Cancer 2014; 50 (suppl 6): 183 (abstr 567).