Recurrent Genomic Aberrations of D-Type Cyclins Are Therapeutic Targets of CDK4/6 Inhibitors in t(8;21) and MLL-Rearranged Acute Myeloid Leukemia

Background:

Acute myeloid leukemia (AML) is a genetically and clinically heterogeneous disease, characterized by expansion of undifferentiated myeloid precursor cells. The outcome for AML has improved through optimal treatment protocols, new drugs, and better supportive care; however, relapse remains common and patients with relapsed AML have poor prognosis. Recent genome-wide analyses revealed several recurrently mutated genes in AML, however, few of these driver mutations have been developed as therapeutic targets to date. In AML, t(8;21) and MLL (KMT2A) rearrangements are among the most frequent chromosomal abnormalities; however, knowledge of the genetic landscape is limited.

Patients and Methods:

The AML-05 study is a Japanese nationwide multi-institutional study of children (age < 18 years old) with de novo AML, conducted by the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG). The trial was registered with the UMIN Clinical Trials Registry (UMIN-CTR; http://www.umin.ac.jp/ctr/index.htm; number UMIN000000511) and conducted in accordance with the principles set down in the Declaration of Helsinki, and approved by the Ethics Committees of all participating institutions. All patients, or their parents / guardians, provided written informed consent. For whole-exome sequencing (WES), whole-exome capture was accomplished by liquid phase hybridization of sonicated genomic DNA using a bait cRNA library (SureSelect Human ALL Exon V5 or V5 Inc RNA 5 kit), following the manufacturer's protocol. Massively parallel sequencing of the captured targets was performed using a HiSeq 2000/2500 (Illumina) with the paired-end 126-133 bp read option. For targeted sequencing, target enrichment was performed using a SureSelect custom kit (Agilent) designed to capture all coding exons of the 338 genes. Similarly, CCND1, CCND2, and CCND3 were also captured and sequenced in t(8;21) AML samples. For cell cycle analysis, cell lines were treated with DMSO, palbociclib (500 nM), or abemaciclib (500 nM) for 24 h. Then, cells were stained with propidium iodide and analyzed using a FACS Canto II flow cytometer (BD Biosciences).

Results and Discussion:

First, we analyzed paired AML tumor and germline samples from nine pediatric MLL-rearranged AML patients by WES. In total, 52 mutations (mean, 5.8 mutations/patient) were identified, including known mutational targets in AML, such as FLT3, BRAF, SETD2, BCORL1, and WT1. Moreover, novel CCND3 mutation was detected in one patient. Next, we analyzed 56 samples from patients with pediatric MLL-rearranged AML enrolled in the JPLSG AML-05 study, using targeted sequencing. We selected 338 genes, among which were previously reported and putative driver genes, including CCND3, for targeted sequencing. We identified eight mutations in CCND3 in five pediatric MLL-rearranged AML patients (8.9%). All mutations were clustered in the PEST domain. Four of the eight mutations were R271fs, which is a known hot-spot mutation in lymphoid malignancies. Mutations of the other D-type cyclins (CCND1, CCND2) have been reported in t(8;21) AML (Leukemia, 2017); therefore, we also searched for mutations in CCND1, CCND2, and CCND3 by targeted sequencing of samples from pediatric t(8;21) AML patients (n=105). CCND1 (n=3, 2.9%) and CCND2 (n=8, 7.6%) mutations were detected; however, no mutations of CCND3 were detected. By contrast, there were no mutations of CCND1 or CCND2 in MLL-rearranged AML (n=56), suggesting that mutations of D-type cyclins exhibit a subtype-specific pattern in AML. A recent study demonstrated that genomic aberrations that activate D-type cyclins are associated with enhanced sensitivity to the CDK4/6 inhibitor (Cancer Cell, 2017), therefore, we also examined the effects of CDK4/6 inhibitors (abemaciclib and palbociclib) on two t(8;21) AML cell lines (Kasumi-1 and SKNO-1) and five MLL-rearrangement AML cell lines (ML-2, MV4-11, MOLM-13, THP-1, and NOMO-1) were analyzed. All cell lines described above exhibited impaired proliferation after treatment with CDK4/6 inhibitors. Furthermore, treatment of these cell lines with CDK4/6 inhibitors resulted in detection of lower frequencies of S/G2/M phase cells by flow cytometry, suggesting that cells were arrested in G1 phase via CDK4/6 inhibition. These data provide further insights into the genetic basis of, and potential therapeutic strategies in t(8;21) and MLL-rearranged acute myeloid leukemia.