Mutant TET2 Allele Dosage Affects Response to 5-Azacitidine in Acute Myeloid Leukemia
Authors: Stoelzel Friedrich , Fordham Sarah Elizabeth , Lin Wei Yu , Blair Helen , Ruhnke Leo , Kunadt Desireé , Mohr Brigitte , Elstob Claire , Allsop Daniel , Nandana Devi , Soura Emmanouela-Niki , Park Catherine , Fadly Mohd , Rahman Thahira , Heidenreich Olaf , Altmann Heidi , Jones Gail , Menne Tobias , Wobus Manja , Jackson Graham , Marr Helen , Onel Kenan , Meggendorfer Manja , Haferlach Torsten , Bornhäuser Martin , Allan James M. 
Published: February 2020

Personalised medicine is predicted to significantly improve outcomes for cancer patients, but implementation requires comprehensive genetic characterisation of malignant cells to identify therapeutically exploitable vulnerabilities. Using an isogenic cell model system with CRISPR-inactivated TET2 in HEL acute myeloid leukemia (AML) cells and an orthotopic mouse xenograft model we demonstrate that mutant TET2 allele dosage significantly affects sensitivity to 5-azacitidine hypomethylating therapy in AML, with biallelic mutation conferring hypersensitivity relative to monoallelic mutation. In the presence of 5-azacitidine, cell clones with biallelic TET2 mutation had significantly lower cloning efficiency (P = 3 x 10-3) and proliferation in liquid culture (P &lt; 1 x 10-4) compared to isogenic clones with monoallelic TET2 mutation. Mixed populations of monoallelic and biallelicTET2 mutated HEL AML cells were transplanted via intrafemoral injection into Rag2−/−Il2rg−/−129×Balb/c mice, and treatment with 5-azacitidine resulted in significant negative in vivo selection against TET2 null cells relative to cells with monoallelic TET2 mutation (P = 4 x 10-4). Methylation analysis revealed the acquisition of an overall hypermethylation phenotype in TET2 null cells and RNA sequencing identified significant down-regulation of ABCB1 transcript, resulting in concomitant pronounced down-regulation of the MDR1 drug efflux transporter at the protein level. RNA sequencing pathway analysis also identified a global effect on ribosome pathway (KEGG pathway ko03010) transcript levels (Padjusted = 0.002), evidenced by down-regulation of numerous RNA polymerase II components in cells with bi-allelic TET2 mutation compared to cells with monoallelic TET2 mutation.
Consistent with our isogenic model data, we characterise biallelic somatic TET2 mutation in a patient with AML that was chemoresistant to anthracycline/cytarabine-based chemotherapy but acutely sensitive to 5-azacitidine, resulting in durable cytomorphological remission. Integration of next generation sequencing, interphase FISH and SNP array analysis of bone marrow at AML presentation, relapse and during remission was used to infer tumour phylogeny which indicated that disease pathogenesis was initiated by a TET2 nonsense mutation (c.2815C&gt;T, Q939*) with subsequent deletion of the second TET2 allele and a NPM1 mutation (c.863_864ins, TCTG) that arose after the acquisition of bi-allelic TET2 mutation. Furthermore, our data demonstrate that 5-azacitidine treatment almost completely eliminated the TET2/NPM1-mutated clone. 5-azacitidine also induced a modest reduction in ancestral pre-leukemic cells carrying bi-allelic TET2 mutation but negative for the NPM1 mutation, although the majority retained viability and re-acquired the ability to differentiate and recapitulate normal haematopoiesis rendering a cytomorphological remission. These observations suggest that bi-allelic TET2 mutation confers sensitivity to the cytotoxic effects of 5-azacitidine, but that the major effect of 5-azacitidine is the induction of phenotypic re-programming.
The frequency of TET2 mutation in primary AML is estimated at 10-20%, with the majority of these being monoallelic. We determined the frequency of TET2 alterations in AML patients presenting with a chromosome 4 abnormality discernible cytogenetically. TET2 copy number and mutational status were determined using high density SNP arrays and gene sequencing, respectively. In a panel of 30 AML cases with a chromosome 4 abnormality, four patients were heterozygous for TET2 mutation (all deletions resulting in reduced copy number) and three patients were homozygous for TET2 mutation (deletion plus base substitution in two cases and homozygous base substitution resulting from uniparental disomy in one case). Furthermore, all seven cases with TET2 mutation were characterised by cytogenetics that included loss or gain of material on chromosome 4. In contrast, only 1 case with a TET2 mutation had a translocation affecting chromosome 4.
In summary, our data argue in favour of using 5-azacitidine in patients with biallelic TET2-mutated AML and demonstrate the importance of considering mutant allele dosage in the implementation of personalised medicine for cancer patients.

Disclosures
Stoelzel: JAZZ Pharmaceuticals: Consultancy; Neovii: Other: Travel funding; Shire: Consultancy, Other: Travel funding. Jackson:Celgene, Amgen, Roche, Janssen, Sanofi: Honoraria. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.