Type: Oral
Session: 503. Clonal Hematopoiesis, Aging, and Inflammation: Causes and Consequences
Hematology Disease Topics & Pathways:
Research, Fundamental Science, CHIP, Genomics, Hematopoiesis, Metabolism, Biological Processes, Molecular biology
We studied erythropoiesis using scRNAseq in bone marrow (BM) from non-CH (n=27), ATM / ATRX (n=8), PPM1D(n=11) and TP53-CH (n=11). With the exception of one patient, >80% of cells analysed in CH samples did not carry a mutation. Erythroid populations in most TP53-CH patients, but not ATM / ATRX nor PPM1D-CH, upregulated HbF (HBG1, HBG2, log2FC 1.6x) compared with non-CH. We confirmed this finding with HPLC quantitation and surmised that it may be associated with erythroid cell stress. Along the erythroid differentiation trajectory, there is a switch from glycolytic to OXPHOS gene expression from megakaryocyte-erythroid progenitor (MEP) onwards, peaking in erythroid precursor (EPre) cells. Expression of OXPHOS and G1S / G2M cell cycle (CC) genes are also strongly positively correlated in non-CH samples. OXPHOS generates reactive oxygen species (ROS), and expression of antioxidant genes (e.g. GPX4, PRDX2, GSTP1) rise to compensate. Compared with non-CH, MEP and EPre in TP53-CH upregulate OXPHOS but had reduced expression of CC genes and reduced expression of GSTP1, suggesting inadequate response to oxidative stress.
To assess function of haematopoietic stem-progenitor cells (HSPCs) in TP53-CH, we performed colony assays with flow-sorted CD34+ HSPCs from 4 TP53-CH samples with different mutations (C238Y, S241Y, R248Q, I255F). The GM-E ratios were no different from controls. However, consistent with reduced expression of CC genes, cloning efficiency was significantly lower in CH than in non-CH controls (mean 21% vs 40%, p<0.05). To investigate mutational effects, we genotyped colonies and found reduced frequency of TP53-mutant (MUTTP53) erythroid colony forming units (CFU-E, reflecting EPre activity) relative to burst-forming units (BFU-E, reflecting MEP activity), indicative of clonal impaired differentiation, in patient #126 (hotspot R248Q, VAF 36%) only. This patient had normal blood counts at index sampling but developed AML 11 months later.
To examine if OXPHOS upregulation is mutant clone-autonomous, and if mutations conferred different biological effects, we compared scRNAseq data in 4 MUTTP53 and their ‘isogenic’ wild-type (WT) patient-derived BFU-E (with cells from erythroid precursors to polychromatic erythroblasts). Only patient #126 showed significantly increased OXPHOS and CC expression in the MUTTP53-R248Q versus its WT control in erythroblasts. MUTTP53-R248Q cells also upregulated thioredoxin (TXN), which scavenges ROS, and mitochondrial uncoupling protein 2 (UCP2) which uncouples OXPHOS from ATP synthesis to reduce ROS. Upregulation of these antioxidant genes may confer metabolic and survival advantage in MUTTP53-R248Q cells over WT counterparts. We predict that this advantage would be greatest in OXPHOS-dependent differentiating progenitors rather than HSC (which are preferentially glycolytic). Indeed, genotyping of HSPCs from #126 shows TP53-R248Q clonal enrichment in CMP, MEP and GMP (VAF 34-38%) versus HSC/MPP (VAF 27%).
In summary, we report that erythroid cells from TP53-CH exhibit signs of decompensated oxidative stress that is not directly driven by mutant clones. We identify metabolic differences between MUT and WT erythropoiesis which may underlie competitiveness in TP53-CH patients. Intriguingly, compared with non-CH, TP53-CH patients are more likely to have metabolic syndrome-related disorders that are associated with oxidative stress which may exert clonal selective pressure.
Disclosures: Hasan: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Carmichael: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Bromage: Bristol Myers Squibb: Research Funding. Suragani: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Mufti: Novartis: Research Funding; BMS/Celgene: Research Funding. Gandhi: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Quek: Bristol Myers Squibb: Research Funding.
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