Clonal Haematopoiesis with TP53 Mutations Exhibits Abnormal Erythropoiesis and Erythroid Metabolic Stress

Clonal Haematopoiesis (CH) with DNA damage repair pathway (DDRp, e.g. ATM, ATRX, PPM1D, TP53) gene mutations carries increased risk of myeloid malignancy. In our cohort of older patients having orthopaedic surgery (n=721, mean age 69 years) we found mutations (VAF≥0.2%) in DDRp in 9% of patients (n=64, ATM/ ATRX n=26, PPM1D n=21, TP53n=17). TP53 mutations were missense, with two cancer hotspots (R248Q and R282W). TP53-CH had larger clones (median VAF 2.9%) compared with other DDRp-CH (median VAF 1.4%, p<0.01). Compared with non-CH (n=366) patients with TP53-CH, but not other DDRp mutations, had significantly elevated MCV (94.7 vs. 92.1, p<0.05) without changes in haemoglobin or RDW.

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 (MUT^TP53) 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 MUT^TP53 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 MUT^TP53-R248Q versus its WT control in erythroblasts. MUT^TP53-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 MUT^TP53-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.

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.