In preclinical models of acute myeloid leukemia with FLT3 internal tandem duplication mutation, AMG 176 in combination with gilteritinib demonstrated the ability to synergistically target the disease.
In preclinical models of acute myeloid leukemia (AML) with FLT3 internal tandem duplication (ITD)– mutation, the selective MCL-1 inhibitor AMG 176 in combination with gilteritinib (Xospata) demonstrated the ability to synergistically target the disease, according to early findings presented during the virtual AACR Annual Meeting 2021.1
Specifically, data showed that there was a strong synergistic effect, with a combination index of <0.3, reported in 2 FLT3-ITD mutated AML cell lines that received the combination. Additionally, AMG 176 plus gilteritinib led to an increase in MCL-1 and BCL-2–like protein 11 (BIM) expression in vitro, a decrease in BIM/MCL-1 interaction, and a significant increase with apoptosis.
In mice that were treated with the combination (n = 10), tumor regression was reported in 92.8% at 22 days after tumor implantation, which included 50% of mice being tumor free. The tumor growth inhibition rate at 22 days following tumor implantation was 73.9% in mice that received gilteritinib alone and 76.1% with AMG 176 alone.
“Our results show that AMG 176 plus gilteritinib treatment was effective and well tolerated in mouse models of AML, which supports further evaluation in a clinical setting,” Chen Xiaoyue, a post-doctoral research fellow in Oncology Research, Amgen, the developer of AMG 176, and coinvestigators wrote in a poster that was presented during the meeting.
The 5-year survival rate for patients with AML is 25%, and FLT3 is the most frequently mutated gene in the malignancy. About 25% and 8% of patients with this abnormality harbor FLT3-ITD mutations and FLT3-tyrosine kinase domain mutations, respectively.
Gilteritinib is a potent, selective oral FLT3 inhibitor that is currently approved by the FDA for the treatment of adult patients with FLT3 mutation—positive relapsed/refractory AML. The approval was based on data from the phase 3 ADMIRAL study (NCT02421939), in which the rate of complete remission (CR) or CR with partial hematologic recovery was 21% (n = 29; 95% CI, 14.5-28.8) with gilteritinib at a median follow-up of 4.6 months.2
However, combination therapy to overcome resistance to gilteritinib in patients with FLT3-mutated AML is warranted.
Furthermore, the study authors expressed that FLT3 mutations are antiapoptotic, and MCL-1, which is an antiapoptotic protein and is said to have a critical role in AML cell survival and drug resistance, is commonly expressed in AML. AMG 176 is defined as a highly potent and selective MCL-1 inhibitor that induces rapid commitment to apoptosis in AML.
In the preclinical study, investigators measured the combination index of AMG 176 plus gilteritinib, conducted an immunoblot experiment, conducted a reverse transcription (RT)-polymerase chain reaction (PCR) assay, an MCL-1:BIM complex immunoassay, evaluated co-immunoprecipitation, generated BIM knockout cell lines, and evaluated the in vivo efficacy for the combination and an in vivo BAK activity test.
For the combination index, MOLM 13 and MV4-11 cells were seeded in 96 well plates at 1000 cell/well in 100 μl media, and cells received AMG 176 at EC70 concentration and gilteritinib at EC50, EC70, and EC90 concentrations. The viability of cells was analyzed 72 hours following therapy through the CellTiter-Glo luminescent cell viability assay, and the combination index was calculated via Calcasyn software. Here, investigators noted that AMG 176 synergizes with gilteritinib in FLT3-ITD–mutant AML cell lines, and BIM knockout was found to significantly reduce the synergistic effect between AMG 176 and gilteritinib.
In the immunoblot experiment, MOLM 13 and MV4-11 cells received gilteritinib at 20 nM for 2, 4, 8, 16, or 24 hours. The cells were washed with phosphate-buffered saline, lysed in a radioimmunoprecipitation buffer on ice, and lysates were pre-cleared via centrifugation at 4 degrees Celsius followed by DCTM protein assay analysis to determine the protein concentration.
In the RT-PCR assay, MOLM 13 and MV4-11 cells received dimethyl sulfoxide (DMSO), 250 nM of AMG 176, 20 nM of gilteritinib, or 20 nM of gilteritinib and 250 nM of AMG 176 for 2 days. The total RNA were extracted via RNeasy Kits, and MCL-1, BCL-2, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels were tested via SYBR Green qPCR.
The MCL-1 and BCL-2 mRNA levels were normalized through GAPDH mRNA levels before being normalized against DMSO treatment control. Data showed that MCL-1 protein expression is increased in samples that received gilteritinib alone or in combination with AMG 176. Additionally, the portion of MCL-1 sequestering BIM significantly decreased in the combination-treated samples vs DMSO control (P <.05).
Additionally, in the preclinical models, gilteritinib was found to increase BIM expression, and overexpressed BIM proteins were sequestered by MCL-1 and BCL-2 following treatment with the FLT3 inhibitor. BIM was released from the BIM/MCL-1 complex after treatment with both drugs; however, compared with gilteritinib monotherapy, the amount of BIM that was sequestered by BCL-2 did not increase with AMG 176 plus gilteritinib.
When generating the BIM knockout cell lines by CRISPR/Cas9 technology, in which MV4-11 cells were sent to Synthego for engineering and 3-guide RNAs that were located at the second exon of the BCL2L11 gene were used, it was found that the BCL2L11 gene can produce multiple BIM isoforms through alternative splicing.
For in vivo efficacy of the combination, 5 x 106 MV4-11 cells were injected into female athymic nude mice. The mice were randomized into 4 groups 12 days following tumor implantation, with 10 mice in each group. AMG 176 was given at 30 mg/kg on a schedule of 2 days on/5 days off for 2 weeks; gilteritinib was given at 3 mg/kg daily. Mouse body weight and flank tumor size were measured twice weekly.
For the in vivo BAK activity, the student’s t-test values were as follows: vehicle vs AMG 176 (P = .0010), vehicle vs gilteritinib (P = .2825), vehicle vs combination (P = .0178), AMG 176 vs combination (P = .2504), and gilteritinib vs combination (P = .0157). No significant decrease in body weight was observed between the 2 groups.
1. Chen X, Caenepeel S, Belmontes B, et al. Efficacy of AMG 176 in combination with gilteritinib in preclinical models of acute myeloid leukemia. Presented at: AACR Annual Meeting 2021; April 10-15, 2021; Virtual. Abstract 1050.
2. FDA Prescribing Information: Xospata (gilteritinib) tablets, for oral use. Accessed April 11, 2021. https://bit.ly/2SjB8CL