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BET and FLT3 inhibition for FLT3-ITD AML
chromatin to control transcriptional activation and cell cycle progression in leukemogenesis.17 Of the four BET family members, BRD4 has been of particular interest in drug development as it directly recruits positive transcrip- tion elongation factor complex b (P-TEFb) to promote tran- scription of MYC and other proto-oncogenes.18
Previous work revealed that the combination of BET inhibition and FLT3 inhibition led to synergistic cytotox- icity in FLT3-ITD AML cells.19 Although this was an important proof-of-principle, these studies were carried out using cells in suspension culture, which does not address the fundamental problem of microenvironment- mediated resistance to FLT3 inhibition. Furthermore, in order to translate this in vitro combination appropriately into the clinic, a BET inhibitor is needed that can achieve adequate MYC suppression in humans. We report here that the combination of PLX51107,20 a structurally novel BET inhibitor currently in early phase clinical studies, and quizartinib, a highly selective FLT3 inhibitor, results in synergistic cytotoxic effects in FLT3-ITD AML blasts on bone marrow (BM) stroma, and that this combination represents a clinically viable strategy to overcome microenvironment-mediated resistance to FLT3 inhibi- tion.
Methods
FLT3 and BET inhibitors
PLX51107 was synthesized at Plexxikon Inc. (Berkeley, CA, USA), a Daiichi Sankyo subsidiary. Quizartinib was obtained from LC Laboratories (Woburn, MA, USA). Stock solutions in dimethyl sulfoxide (DMSO) were stored at -20°C. Dilutions from the stock solutions were prepared in RPMI (Gibco, Waltham, MA, USA) with 10% fetal bovine serum (FBS) (Gemini Bio Products, Sacramento, CA, USA), penicillin/strepto- mycin, and 2 M L-glutamine (Gibco, Waltham, MA, USA). The final concentration of DMSO in all experiments was ≤0.1%.
Patients’ samples
Patients' leukemia samples, as well as plasma and BM from healthy donors, were acquired under an institutional review board-approved protocol from the Johns Hopkins Tumor and Cell Procurement Bank. Patients gave informed consent accord- ing to the Declaration of Helsinki.
Cell cultures
Cell lines and primary leukemia cells were cultured in RPMI with 10% FBS, penicillin/streptomycin, and L-glutamine at 37°C in 5% CO2 OCI-AML3 (expressing a wild-type FLT3 gene, an NPM1 gene mutation [type A], and a DNMT3A R882C muta- tion) and Molm14 cells (expressing a 21 bp FLT3-ITD mutation) were purchased from Deutsche Sammlung von Mikroorganismen and Zellkulturen, Braunschweig, Germany. MV4-11 cells (FLT3-ITD mutated) were from American Type Culture Collection, Manassas, VA, USA.
Cytotoxicity assays
Primary co-cultures in 96-well plates were incubated with specified drug treatments for 72 hours (h) prior to the assess- ment of cytotoxicity by a dimethyl-thiazole diphenol tetrazoli- um bromide (MTT) assay (Roche Diagnostics, Indianapolis, IN, USA) as described.21 Experimental duplicates were also concur- rently counted for cell viability by trypan blue exclusion under light microscopy.
Immunoblotting
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The antibodies to probe for MYC and β-actin were obtained from Cell Signaling Technologies (#5605, Danvers, MA, USA) and β-actin (13E5, Danvers, MA, USA).
Plasma inhibitory activity assays
Plasma for the plasma inhibitory activity (PIA) assays was col- lected from patients enrolled in a phase Ib/IIa dose- escalation/expansion study of PLX51107 (clinicaltrials.gov identifier: NCT02683395). Plasma samples were collected from cycle 1 on day(d)1at0,0.5,1,2,3,5,7,and9h,andpre-doseond2(i.e.,24 h post d1 dose). The samples were stored at -80°C and used with- in 12 months of collection for the PIA assays.
The PIA assay was adapted from a previously described approach.22 OCI-AML3 cells were incubated with each plasma sample for 3 h at 37oC in microcentrifuge tubes. Each OCI-AML3 sample was then centrifuged and washed a total of five times each in phosphate buffered saline (PBS) prior to cell lysis. Lysates were cleared by centrifugation. 50 mg of lysates were subjected to sodi- um dodecyl sulphate-polyacrylamide gel electrophoresis (SDS- PAGE) and immunoblot, as described above. Membranes were probed for antibodies against MYC and β-actin, as described above.
Colony-forming assays
Colony-forming assays of normal human BM progenitors were performed as described.23
Information concerning primary stromal culture and leukemic cell co-culture, xenograft studies, RNA sequencing and quantita- tive-polymerase chain reaction (PCR) is available in the Online Supplementary Methods.
Results
Previous work demonstrated a synergistic cytotoxic effect of the BET inhibitor JQ1 and FLT3 inhibition in FLT3-ITD AML cells in suspension culture.19 To pursue this concept in vivo, we first tested it in a mouse model of FLT3-ITD AML. JQ1 is poorly suited to in vivo application. However, PLX51107 is a structurally distinct BET inhibitor that exhibits low nanomolar affinity and a unique binding position,20 and is rationally designed for therapeutic use in humans. PLX51107, which has no activity as a FLT3 inhibitor (Online Supplementary Figure S1), potently inhib- ited the in vitro growth of two AML cell lines harboring FLT3 ITD mutations, MV4-11 and MOLM-14, with IC50 values of 62 and 79 nM, respectively (Online Supplementary Figure S2), suggesting that these cell lines are susceptible to BET inhibition. Despite its short plasma half-life, oral administration of PLX51107 led to significant transcrip- tional changes of a BET target gene HEXIM1 in MV4-11 cells xenografted in female SCID mice, confirming target engagement in vivo.20 Here we used the same model but extended the duration of the study to examine the in vivo- efficacy of PLX51107 as a single agent or in combination with quizartinib in treating FLT3-ITD AML.
We first evaluated three oral doses (10, 20 and 40 mg/kg daily) of PLX51107 for efficacy in blocking MV4-11 xenograft growth. PLX51107 treatments demonstrated dose-dependent suppression of tumor growth (Figure 1A), achieving >50% tumor growth inhibition at 10 mg/kg dose (corresponding AUC0-24 = 29,300 ng x h/mL) and above. PLX51107 dosed at 40 mg/kg resulted in tumor regression between d4 and d13 of dosing. By d14 (the last
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