Type: Oral
Session: 631 Myeloproliferative Syndromes and Chronic Myeloid Leukemia: Basic and Translational: Oncogenic Drivers and Genetic Models
Hematology Disease Topics & Pathways:
Research, Translational Research, Diseases, Myeloid Malignancies
To characterise the contribution of structural variants to BPMPN, we first performed integrated copy number and mutation profiling in 64 BPMPN patients by SNP karyotyping and targeted next generation sequencing. We observed a recurrent pattern of chromothripsis that involved chromosome 21, which together with other structural variants led to amplification of a common region of chromosome 21 (‘chr21amp’) in ~25% of patients (GISTIC q-val<0.01, Fig 1A). Chr21amp was associated with TP53 mutations and a higher number of copy number alterations. Patients with chr21amp had a particularly aggressive and treatment-resistant phenotype, with 0% surviving 12 months compared to 46% in the non-chr21amp pts (p=0.0007), retaining significance in multivariate analysis including after correction for TP53 mutation status.
Whole genome sequencing confirmed that the chromosomal rearrangements resulting in chr21amp occurred by different mechanisms, ranging from simple amplification to highly complex chromothriptic events involving multiple chromosomes. There were no recurrent translocation partners or mutations. The minimally amplified region (MAR) spanned 2.7Mb and contained 24 genes, with a median copy number of 3.5 (range 2.7-8.3)
Single-cell transcriptomics combined with allelic resolution genotyping revealed that chr21amp was present in the dominant subclone and occurred subsequent to JAK2V617F and mutTP53 acquisition. Chr21amp was detectable in phenotypic HSCs and throughout early stages of hematopoiesis, but not in mature erythroid cells, consistent with a differentiation block.
Of the 24 genes in the minimally amplified region, only one gene, DYRK1A, a serine threonine kinase linked to cell proliferation and survival, was both differentially expressed (single-cell and bulk RNAseq) and differentially accessible (ATACseq). To explore the functional role of DYRK1A in BPMPN we performed shRNA and CRISPR-mediated DYRK1A-knockdown and knockout (KO) in BPMPN cell lines (HEL and SET-2), which led to impaired cell proliferation. The DYRK1A inhibitors EHT1610 and GNF2133 also led to dose-dependent growth inhibition. DYRK1A-KO BPMPN HEL or SET2 cell clones showed a reduced ability to propagate leukemia in vivo with a significant survival advantage vs. wild type control mice. BPMPN chr21amp+ primary patient CD34+ cells were highly sensitive to DYRK1A inhibitors, while healthy control CD34+ cells were unaffected
Prior studies have shown that DYRK1A activates the DREAM complex, a transcriptional repressor of DNA-repair pathways. In chr21amp patient cells, the DREAM DNA repair gene signature was significantly downregulated (NES -1.74, q-val <0.001), while conversely in CRISPR DYRK1A KO SET2 cells the transcriptional DNA repair signature was upregulated (NES 1.76, q-val <0.001). In functional assays, CRISPR DYRK1A KO was protective against DNA damage, with a reduction in 𝛾-H2AX foci after etoposide treatment or irradiation (p<0.01 for both).
Disclosures: Wen: AstraZeneca: Current Employment. Levine: Isabl Inc.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. O Sullivan: Novartis: Honoraria; Morphosys: Honoraria. Harrison: AOP: Honoraria, Speakers Bureau; CTI: Honoraria, Speakers Bureau; Galecto: Honoraria, Speakers Bureau; Morphosys: Honoraria, Speakers Bureau; Abbvie: Honoraria, Speakers Bureau; GSK: Honoraria, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Speakers Bureau. Drummond: Novartis: Other: Personal fees,, Research Funding; Blueprint Medicines Corporation: Research Funding. Knapper: Jazz: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Tolero: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Daiichi Sankyo: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees. Constantinescu: MyeloPro Diagnostics and Research GmbH Vienna: Other: Co-founder; Novartis: Speakers Bureau; GSK Belgium: Membership on an entity's Board of Directors or advisory committees. Papaemmanuil: TenSixteen Bio: Current equity holder in private company; Isabl Inc.: Current equity holder in private company, Current holder of stock options in a privately-held company, Other: CEO, Patents & Royalties: Whole genome cancer analysis. Psaila: GSK: Honoraria; Blueprint Medicines: Honoraria; University of Oxford: Patents & Royalties: 2203947.3 ; Novartis: Speakers Bureau. Crispino: SAB of Alethiomics: Other: Member; Cellarity: Consultancy. Mead: Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; CTI: Consultancy, Speakers Bureau; Galecto: Consultancy, Research Funding, Speakers Bureau; Alethiomics Ltd: Consultancy, Current equity holder in private company, Other: Cofounder & equity holder, Research Funding; Incyte: Consultancy, Speakers Bureau; Karyopharm: Consultancy, Speakers Bureau; Sierra Oncology: Consultancy, Speakers Bureau; Sensyn: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; University of Oxford: Patents & Royalties: 2203947.3 ; Celgene/BMS: Consultancy, Research Funding, Speakers Bureau; AbbVie: Consultancy, Other: investigator for AbbVie sponsored trials, Speakers Bureau; Relay Therapeutics: Consultancy, Speakers Bureau; GSK: Consultancy, Speakers Bureau; Roche: Research Funding.