Long-Term Clonal Inversion in an MDS-RS Case with Dual SF3B1 Mutations

Recent large-scale studies have traced SF3B1 mutations as a driving event in clonal establishment and expansion, and a hallmark of myelodysplastic syndrome with ring sideroblasts (MDS-RS). However, the mechanisms underlying the fitness advantage allowing SF3B1-mutant (SF3B1^mt) clones to thrive remain poorly understood. Thus, tracking these events in conditions of low noise is crucial to our comprehension of clonal hematopoiesis.

Here we report a 12-year clinical and molecular follow-up of a unique dual SF3B1^mt MDS-RS case displaying a dynamic state of clonal competition between two clones propagated by a N626D and K666N mutation, respectively (Panel A). After an initial rapid expansion of the N626D clone compared to the relatively stable K666N clone during the initial follow-up period of ~2 years, the K666N clone spontaneously expanded at expense of the N626D clone to eventually dominate the bone marrow, driving a complete inversion of clonal prevalence. Clinically, these active shifts of high clonal involvement in the bone marrow were accompanied by stable subclinical anemia (median 12.5 g/L, range 12.0 - 13.2 g/L) and a continued decrease in the ring sideroblast phenotype over time (from 42% to 8% RS) in the absence of any treatment. The stability of this patient's clinical profile was therefore considered as unlikely to confound further characterization.

Through a combination of whole-genome sequencing (WGS) of hematopoietic stem and progenitor cell (HSPC)-generated colony-forming units (CFU), combined DNA + RNAseq (TargetSeq) of FACS-purified hematopoietic stem cells (HSCs) and megakaryocyte-erythroid progenitor cells (MEP), and high-throughput 3'-based 10X Genomics single-cell RNAseq of CD34⁺ HSPCs, we molecularly characterized the two distinct SF3B1^mt clones in bone marrow samples acquired before, during and after the points of clonal inversion (Panel B).

Using WGS of single cell-derived colonies, we established that the acquisition of either the N626D or K666N mutation had preceded the diagnosis of MDS-RS by at least 20 years. Moreover, both mutations were associated with an increased mutational rate as compared to wildtype colonies, and the two clones presented distinct patterns of additional coding mutations as potential co-drivers of expansion.

Mirroring previous characterizations of SF3B1^mt HSPCs, we identified a common and deleterious alternative splicing profile to both genotypes via TargetSeq which included mis-splicing events in the known SF3B1^mt targets MAP3K7 and SEPTIN6. In addition, we identified mutation-specific splicing profiles as potentially responsible for distinct downstream distinctions between both clones. Through this analysis, we identified the K666N-specific loss of expression of the tumor suppressors HOXA5, RGCC and NEURL1 as potential compound factors to the known higher risk underlying SF3B1^K666N mutations. Finally, we disentangled clone-driven and aging-driven transcriptomic changes to identify a significant decrease of the early hematopoietic controllers ID1, ID2 and ID3 as a potential mechanism of aging-related cellular senescence.

In conclusion, this case study comprises a unique long-term follow-up of the clonal dynamics underlying two co-existing, distinct and competing SF3B1^mt clones which identifies subtle molecular changes and differences underlying the expansion and progression of SF3B1^mt clones.

*Moura PL, Hofman IJF, Nannya Y and Aliouat A contributed equally to this work.

Nakagawa:Sumitomo Dainippon Pharma Oncology, Inc.: Research Funding. Ogawa:15/353395 (US03): Patents & Royalties; PCT/JP2014/062112 (WO01): Patents & Royalties; 2013-526957 (JP02): Patents & Royalties; DaiichiSankyo: Speakers Bureau; Pfaizer: Speakers Bureau; ASAHI Genomics: Current equity holder in publicly-traded company; 2014-191287: Patents & Royalties; Novartis: Honoraria, Speakers Bureau; The Mitsubishi foundation: Honoraria; MSD: Speakers Bureau; Clinical Research Support Center Kyushu: Research Funding; 62/187386 (US01): Patents & Royalties; Otsuka Pharmatheutical: Research Funding; Kirin/Chugai: Speakers Bureau; Astellas: Speakers Bureau; The Chemo-Sero-Therapeutic Research Institute: Speakers Bureau; Chordia Threapeutics: Consultancy, Current equity holder in publicly-traded company, Research Funding; Astrazeneca: Speakers Bureau; Sysmex: Honoraria; Nanpu Hospital: Research Funding; Esai Pharmatheutical: Consultancy; 2013-096582 (JP01): Patents & Royalties; 2015-239547: Patents & Royalties.