The basis for the differential impact of BCOR and BCORL1 in aplastic anemia/paroxysmal nocturnal hemoglobinuria and myeloid neoplasia Free

and BCORL1 (BCOR/L1) are X-chromosomalTSGs affected by inactivating or hypomorphic somatic mutations (^MT) in myeloid neoplasia (MN). However, BCOR/L1^MT are also present in the context of immune-mediated bone marrow failure, including aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH) as clonal hematopoiesis. While BCOR^MT appears to be a poor prognostic factor in MN, in AA/PNH, it represents a self-limited clonal expansion with carriers having a lower risk of secondary MN progression¹. This discordant impact of BCOR/L1^MT constitutes a clinical conundrum, which can be deconstructed into 2 main pathogenetic issues: i) differential leukemogenic properties in AA/PNH and MN with their disconnection from the secondary MN evolving from prior AA/PNH; ii) immunologically privileged factors leading to the preferential expansion of otherwise benign BCOR/L1^MT clones in AA/PNH. There are possible theories addressing these issues including; a) decreased pathogenicity of BCOR/L1 hits in AA/PNH due to the higher proportion of missense mutations and/or their predominance in AA/PNH females; b) occurrence without a context of somatic founder mutations. Either way, preferential expansion of BCOR/L1^MT clones may be predicated by extrinsic factors operative only in the context of AA/PNH milieu. They may involve abundance of hematopoietic growth factors, immune attack selecting for phenocopy of PNH due to lack of glycosylphosphatidylinositol-anchored proteins (GPI-APs), or deficient fitness/quantity of remaining stem cells. The latter factors may facilitate somatic gene rescue via the acquisition of BCOR/L1^MTmutant clones.

To gain more insight into this, we started by analyzing the mutational context of BCOR/L1^MT. In 10,332 patients (institutional, 3328; public series, 7004), somatic 372 BCOR^MT (3.6%) and 66 BCORL1^MT (0.6%)were found, including 42 cases with bi-clonal mosaicism. We found similar mRNA expression in males and females, indicating almost complete lyonization. BCOR/L1^MT rates were comparable between MN (n=9,414; 5%), AA/PNH (n=918; 4%), and CHIP/CCUS (n=212, 6%). In AML, single-cell NGS data suggested that BCOR is subclonal, confirming its role of a secondary leukemogenic driver². In AA/PNH, PNH clones were significantly more frequent (96% vs 72.1%, P=.013) in BCOR/L1^MTpatients.

Next, we assessed the differences between AA/PNH and MN. Patients with BCOR/L1^MT in MN were significantly older than in AA/PNH (P<0.001), occurring at the same proportion in old vs. younger AA patients. BCOR/L1^MT carriers in AA/PNH were characterized by a higher prevalence of missense and in-frame indels (P=.036) and a significantly smaller clonal burden compared to AML (P<.001). In AA/PNH, no BCOR/L1^MT patients coincided with somatic HLA mutations and locus deletions, and the distribution between female and male BCOR/L1^MT patients was not significantly higher to that of MN (53% vs 36%, P=.122). In AA/PNH, BCOR/L1^MT predominantly occurred alone (n=38; 53%) or as clonal mosaicism with PIGA^MT (21%). In contrast, in MN, BCOR/L1^MT frequently co-occurred in the same clone with RUNX1^MT, DNMT3A^MT, and ASXL1^MT.

We also studied potential factors promoting BCOR expansion using RNA-seq. Following normalization based on cell composition using CIBERSORTX, RNA-seq of 644 immune effector and GPI-linked proteins demonstrated that, BCOR^MT AA exhibited decreased expression of ICOSLG, CD274, and HPSE but no difference in checkpoint molecules, likePD1, TIM2, and VISTA. The mRNA levels of HLA and β2microglobulin did not differ between BCOR/L1^MT and WTcases. However, K562 BCOR^KO did show HLA class I, downmodulation to isogenic wildtype. Interestingly, BCOR/L1 expression in AA/PNH was significantly reduced compared to controls (P<.001). We also investigated whether BCOR/L1^MT results in a phenocopy of PNH by downregulation of GPI-APs. Indeed, certain GPI-APs were downregulated in BCOR/L1^MT AA including e.g., CD14, HPSE, ENTPD2, and PRDM12, suggesting that some GPI-APs may partially phenocopy PNH involved in immune advantage in the context of immune attack.

In conclusion, while clear differences in the genetic context of BCOR/L1 were found compared to MN, we could not definitely establish a reason for the expansion of BCOR/L1^MT clones in the AA/PNH environment. However, downmodulation of certain GPI-APs may resemble the PNH phenotype, and indeed, these two genetically distinct clones may share functional similarities in the context of AA/PNH.

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