Sirenko, Maria

ABSTRACT Hypomethylating agents (HMA) and allogeneic hematopoietic stem cell transplantation (alloHSCT) have both demonstrated remissions in VEXAS; however, comparative data is lacking. We conducted a multicenter, retrospective analysis of 66 patients diagnosed with VEXAS syndrome treated with HMA ( n = 35) or alloHSCT ( n = 31). Baseline characteristics such as genetics, co‐morbidities, and performance status were balanced between the groups, except older age in the HMA group. Median follow‐up from therapy initiation was 18 months (95% CI: 11–26), and 14 (21%) deaths were reported (alloHSCT n = 3; HMA n = 11). Among all evaluable patients within the alloHSCT cohort, all patients achieved molecular remission, and a substantial proportion of patients discontinued glucocorticoids (58%). In contrast, HMA therapy was associated with lower but meaningful rates of molecular remission (22%) and glucocorticoid discontinuation (6%). In a real‐world setting, HMA therapy was associated with a high discontinuation rate related to toxicity or lack of response. On multivariable analysis adjusted for age and Charlson Comorbidity Index, alloHSCT was associated with improved overall survival (HR = 0.20, 95% CI: 0.05–0.81; p = 0.024). This association remained consistent across multiple ancillary sensitivity analyses, including restriction to transplant‐eligible patients, patients aged ≤ 75 years, 1:1 matching, and propensity score‐based weighted analyses. Although limited by retrospective design, these findings suggest that alloHSCT remains an attractive and potentially curative strategy in selected patients with VEXAS. Prospective validation of these findings is warranted.

Abstract Introduction: Somatic mutations in UBA1, encoding an E1 ubiquitin activating enzyme, have recently been linked to an adult-onset, severe inflammation syndrome called VEXAS (Vacuoles, E1-enzyme, X-linked, Autoinflammatory, Somatic). VEXAS is characterized by systemic inflammation and hematologic abnormalities. While the majority of identified mutations in UBA1 are located in exonic regions, the role of non-coding and intronic mutations in the pathogenesis of VEXAS remains largely unexplored. In this study, we investigated the potential contribution of intronic mutations to VEXAS pathogenesis. Methods: To investigate mutations affecting UBA1 outside the region typically covered in targeted panels (exon 3), whole exome (WES) and genome sequencing (WGS) was performed on peripheral blood of patients with VEXAS-associated clinical presentation previously negative for UBA1 mutations per clinical testing. 265 patients were profiled by WES (~100X) and 36 patients were profiled by WGS (~55X). Clinical history was reviewed for these patients for VEXAS-associated presentation including a combination of hematologic and rheumatologic disease in adults. Participants with known pathogenic or likely pathogenic (P/LP) variants in UBA1 or other genetic causes of systemic autoinflammatory disease were excluded. UBA1 transcript and protein expression were assessed by PCR and western blot, respectively, in one case where peripheral blood mononuclear cells were available. Results: We identified 7 patients with VEXAS-like presentation with intronic single nucleotide variants or deletions. Five patients had a recurrent point mutation (median VAF: 0.78; range: 0.38-0.97). 5 patients had been diagnosed with MDS, and bone marrow biopsy revealed vacuolated progenitor cells in 3 patients. The most frequent clinical manifestations included skin rash (n=6), joint pain (n=4), vasculitis (n=4), and chondritis (n=2). One patient had been diagnosed with acute myeloid leukemia with myelodysplasia related changes, with co-mutations in IDH1 and SRSF2. Two additional patients had mutations in ASXL1 of which one also had a ZRSR2 mutation. No co-occurring myeloid driver gene mutations were found in the other cases. Noncoding mutations, similar to canonical UBA1 disease causing mutations, were shown to be lineage restricted to myeloid cells. To assess the functional impact of mutations, CD14+ monocytes were isolated from the peripheral blood. Compared to the CD14- fraction, the CD14+ fraction revealed a decrease in the UBA1 transcript as well as UBA1 protein level. One patient demonstrated loss of novel UBA1 mutation after hypomethylating agent treatment, and allogeneic transplant, placing the patient in remission.Conclusions:Our findings of intronic and non-coding UBA1 mutations in patients with VEXAS-associated clinical presentation suggests these variants may affect the regulation of UBA1 transcription, representing a potential new mechanism of pathogenesis. Further functional studies are underway to understand how these variants contribute to impaired UBA1 activity and VEXAS disease pathogenesis. Collectively, our results advocate for a broader approach in genetic testing for UBA1 mutations and pave the way for improved diagnostic and therapeutic strategies for VEXAS syndrome.

Abstract Introduction: VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) is an adult-onset autoinflammatory disorder defined by somatic UBA1 mutations in hematopoietic stem cells (HSCs). Given the higher median age at onset and prevalent inflammation, approximately 60% of patients have concurrent clonal hematopoiesis (CH) involving epigenetic regulators, DNMT3A and TET2. We have previously demonstrated (Gutierrez-Rodrigues, Blood 2023) that DNMT3A and TET2 mutations in VEXAS are independently associated with increased mortality, without an increased risk of myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). To define the role of CH in VEXAS, we investigated the impact of CH on mortality and related epigenetic dysregulation in primary VEXAS patient samples and validated these findings using an adenine base-editing (ABE) strategy to model cooccurring UBA1 and DNMT3A mutations in CD34+ HSCs. Methods: After IRB approval, 114 VEXAS patients from the Mayo Clinic and the NIH were retrospectively assessed, including 104 screened for CH by error-corrected DNA sequencing (ECS). DNA methylation changes were identified using the Illumina Infinium MethylationEPIC BeadChip and genome-wide using Biomodal 6-base sequencing on peripheral blood samples (PB). Cellular indexing of transcriptomes and epitopes (CITE)-seq was performed using the 10x Chromium 3' GEX platform; 21,114 pooled cells were subjected to UMAP dimensionality reduction, followed by Azimuth based cell identification. HSCs from different donors were base edited for UBA1 M41T mutation with and without cooccurring DNMT3A splice-site mutations. Levels of cytokines in cell culture supernatants were measured using Olink technology, transcriptomic changes were assessed using bulk RNA-seq. Statistical analyses were carried out using R. Results: Patients' median age was 68 (62-74) years; 95% were white and 99% were males. 39% had a pathogenic CH variant, with DNMT3A (18%; median VAF: 42%; range 6-46%) and TET2 (11.5%; median VAF: 11%; range: 2-37%) being most common (2% had both). While the presence of any CH mutation did not impact survival (7.7 vs 11.5yrs, p =0.69), DNMT3Amt CH had an age-independent negative impact on mortality (9.2 vs 5.6yrs, p=0.02), without an increased risk of MDS/AML (p=0.31). Changes in DNA methylation were profiled using the methylation array in 19 VEXAS patients without CH (Median Age: 68 years) and in 9 with DNMT3Amt CH (Median Age: 74 years), respectively. VEXAS samples with DNMT3Amt CH displayed a hypomethylation phenotype compared to VEXAS samples with no CH, with 4,096 unique hypomethylated CpGs, occurring mainly at gene enhancers (Enh) and transcriptional states (Tx, TxWk). This loss of DNA methylation was enriched in inflammatory pathways with extensive hypomethylation observed in the promoters and gene bodies of IL-1 family genes. CITE-seq performed on 7 VEXAS samples (4 DNMT3Amt CH vs 3 no CH) demonstrated an upregulation of gene expression predominantly in T-cell subsets. This increased expression was enriched in inflammatory genes, largely centered around IL-1 and IL-18 family signaling. Pathway analysis revealed an enrichment in respiratory electron transport and oxidative phosphorylation (oxphos), with the VEXAS samples with DNMT3Amt CH demonstrating significantly increased oxphos gene signatures. Base editing of CD34+ cells was done using an adenine base-editing (ABE) strategy to model dual UBA1 and DNMT3A splice-site mutations. We observed that the HSCs base edited for the UBA1 M41T recapitulated the cellular hallmarks of VEXAS demonstrating reduced ubiquitination, unfolded protein response (UPR) activation, and increased inflammation. The addition of cooccurring DNMT3A mutations resulted in decreased DNMT3A expression, further increasing inflammatory markers, confirming altered inflammatory and metabolic pathways. Conclusion Our study validates the age-independent, negative prognostic impact of DNMT3Amt CH on survival in VEXAS syndrome. DNMT3A mutations mediate epigenetic and transcriptional priming associated with upregulation of inflammatory and mitochondrial function pathways. These findings have been validated in a base edited cell model with UBA1 and DNMT3A co-mutations.

Abstract Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) have a propensity to transform to an accelerated or blast phase (MPN-AP/BP). The resulting disease has clinically similar manifestations to Acute Myeloid Leukemia (AML) but worse clinical outcomes. Here we present the first comprehensive description of the transcriptomic characteristics of MPN-AP/BP. Our analysis incorporates data from 261 patients of the BeatAML cohort and 56 MPN-AP/BP patients, 11 of whom had paired samples from before and after transformation. We establish that transformed MPN is a transcriptionally distinct entity from de novo AML and chronic phase MPNs. Genomic pathways traditionally associated with MPN pathogenesis, such as IL2/STAT5 signaling and IL6/JAK/STAT3 signaling were enriched in chronic-phase MPNs but are absent in transformed disease, suggesting JAK2 directed therapy may be less effective in this disease phase. We also discovered that gene expression signatures associated with doxorubicin resistance are highly enriched in transformed MPNs, which may explain the lack of efficacy of standard AML therapies. In addition, we identify that lineage composition at the time of transformation may define distinct subsets of MPN-AP/BP patients, which may assist in the future development of novel treatment strategies.