Citations

Abstract The importance of bone marrow (BM) microenvironment and its immune interactions within and their role in chronic myelomonocytic leukemia (CMML) disease progression have not been well studied. Our previous work in CMML identified type 1 helper T cell (Th1) and CD4 terminal effector cell suppression in peripheral blood from untreated patients, in comparison to age-matched normal controls (Mangaonkar A et al. Blood Adv 2020). Furthermore, clonal dendritic cell (DC) aggregates in CMML are present in approximately 20-30% patients at diagnosis, display significant cell heterogeneity (plasmacytoid DCs + myeloid DCs and other myeloid/immune cells), express indoleamine 2,3-dioxygenase-1 (IDO), associate with T regulatory cell (Treg) expansion, and transformation to AML [blast transformation or BT, Lucas N et al. Leukemia 2019, Mangaonkar A et al. Blood Adv 2020, and Mangaonkar A et al. Blood 2023 (abstract)]. The purpose of this study was to define the myeloid-immune cell interactions within the BM microenvironment critical for CMML progression to AML. To phenotypically identify cell types and their interactions within the CMML BM, we used single-cell type spatial omics through two distinct modalities on archival formalin-fixed paraffin-embedded (FFPE) BM biopsy specimens. Ten serial CMML samples at chronic phase [CP, n=5, median BM blast% 5 (2-15)] and at BT [n=5, median BM blast% 60 (25-74)] were used for both techniques. Spatial transcriptomics was conducted through GeoMx Digital Spatial Profiler (DSP) platform that uses UV-photocleavable, spatial-barcoded oligo probes to profile the whole transcriptome in defined regions of interest (ROI). CD123 (marker for DC-myeloid aggregates) immunofluorescence antibody was used to identify and segment ROI. 16-color flow cytometry was done to validate cell-specific expression. Spatial cellular phenotyping of immune cells on the same samples (serial cuts) was done through multiplex imaging by co-detection by indexing (CODEX) using 27 markers: CD31, Ki67, CD11b, CD20, CD44, Ecad, CD123, CD107a, CD8, CD11c, CD45RO, FoxP3, CD14, PDL1, CD56, CD68, CD163, PD-1, CD38, HLA-DR, CD4, IDO, CD45, PanCK, Na/K/ATPase, CD3e, and DAPI. CD8T+ cell anergy was demonstrated through CFSE cell proliferation assay. Benjamini-Hochberg correction was used to adjust for multiple hypotheses. After filtering poor quality ROIs [ROIs with total read count < 1000 were removed, ROIs with trim, stitch, alignment < 80% or saturation < 50% were removed], 3rd-quartile normalization [BiocGenerics (v. 0.48.1)] and batch correction [limma (v. 3.58.1)], we compared transcriptomics signatures between CD123+ and CD123- ROIs in all samples. Results showed that LIPA, SLC4A1, HBA2, TIMD4 and HMOX1 werethe top five genes upregulated in CD123+ areas. Among all gene transcripts, TIMD4 expression increased the most (Log2FC=0.6, P-value=8.79E-10, FC=1.52, FDR=1.68E-07) when CD123+ ROIs were compared between CMML-CP and CMML-BT serial samples, suggesting a biological relevance in disease progression. Previous work in metastatic peritoneal cancer has established that TIM-4 expressing macrophages suppress Th1/Th2 ratios and impair CD8+ T cell function (Chow A et al. Cancer Cell 2021). To confirm expression at the protein level, we used a multicolor flow cytometry panel and confirmed that TIM4 is expressed on macrophages (Lineage-negative, HLA-DR+, CD68+ cells) in independent CMML BM aspirate samples (n=3). Next, we conducted spatial phenotyping using CODEX on serial cuts from the same serial samples used for spatial transcriptomics. Anatomical co-localization of macrophages with leukemic cells, monocytes and T cells was visually inspected and confirmed. Cell-interaction analysis (after Benjamini-Hochberg correction) indicated that macrophages-monocyte, macrophage-leukemic cells, and macrophage-memory CD4+T cells were significantly (P<0.05) increased in CMML-BT versus CMML-CP. CD8T+ cell anergy was confirmed through a CFSE cell proliferation assay in untreated CMML PBMC samples. In conclusion, our data suggests that TIM4-expressing macrophages and their interactions with monocytes/monocyte precursors and immune cells within a DC aggregate create a protective leukemic stem cell niche and T cell tolerance, promoting blast transformation in CMML.

Abstract Introduction: Somatic mutations in splicing factors (SF) - SF3B1, SRSF2, U2AF1, and ZRSR2 – represent one of the most common drivers of premalignant clonal hematopoesis. While SF mutations are potent drivers of clonal expansion (Fabre et al., Nature 2022), multiple SF mutations are rarely observed in the same individual (negative epistasis). Prior studies have identified rare cases of concurrent SF mutations (<4%) and described specific hotspot combinations that escape negative epistasis (Taylor et al., Blood 2020). We conducted this study to further characterize the frequency and clonal relationship between concurrent SF mutations using a larger patient cohort. Methods: After IRB approval, we assessed 3,369 myeloid next generation sequencing (NGS) results that identified somatic variants in SF3B1, SRSF2, U2AF1, or ZRSR2, in 2,500 adult patients from 08/2015 to 07/2025. Patient demographics, ICD-9/10 codes, and complete blood counts with differentials were obtained, retaining only those datapoints assigned 30 days before or after NGS testing. To allow comparison of clone size between autosomal and the X-linked SF ZRSR2, VAFs were multiplied by two to estimate the cancer cell fraction (CCF), except for ZRSR2 mutations in males. For patients harboring concurrent SF mutations, we categorized them as co-dominant if the difference in respective CCFs was < 10% of the greater CCF. Specific SF variants were categorized into common hotspots (n=13), except for ZRSR2 variants, which do not occur in defined hotspots. Fisher's exact testing was used to assess the enrichment of SF hotspot variants in patients with concurrent SF mutations, as well as combinations of SF variants in patients with co-dominant SF mutations. Results: We identified 2,331 patients with at least 1 pathogenic SF mutation, of which 6.7% (n=156) had > 1 SF mutation (multi-SF MT). The total cohort comprised cases of AML (n=549, 24%), MDS (n=977, 42%), MDS/MPN (n=233, 10%), MPN (n=228,10%), and uncategorized ICD codes (“Other”, n=326), of which the largest category was clonal cytopenia(s). Notably, the prevalence of multi-SF cases was higher than previously described and our cohort included 5 patients with 3 SF mutations and 1 with 4 SF mutations. Of the multi-SF MT patients, we identified 42 (27%) with co-dominant CCFs among their respective SF mutations. Thirty-three multi-SF MT patients had multiple NGS sampling timepoints (median 2, range 2-4) spanning a median of 0.97 years (range 0.06-5.69 years); we found that 17 of these 33 demonstrated correlative SF CCFs over multiple timepoints, suggesting their SF mutations occurred in the same clone (“co-clonal”). Of these 17 patients, 82% had co-dominant CCFs at the single-timepoint level, whereas none of the 16 non-co-clonal demonstrated co-dominance. Thus, we used SF co-dominance as a surrogate for cases likely to be co-clonal. ZRSR2 (p=8.8x10-29), U2AF1Q157 (p=9.4x10-9), SF3B1K666 (p=1.4x10-4) and infrequent SF3B1 (“SF3B1-Other”, p=.0029) mutations were overrepresented in the multi-SF MT cohort compared to patients with a single SF mutation. Within the multi-SF MT cohort, 3 SF mutation combinations showed overrepresentation in codominant cases – SRSF2P95R/H/L+ ZRSR2 (p=0.014), SRSF2P95R/H/L+ SF3B1K666 (p=0.044), and U2AF1S34 + U2AF1Q157 (p=0.024), although not significant after multiple hypothesis correction. Dual ZRSR2 mutations were the most common combination seen in the multi-SF MT cohort but were underrepresented in codominant cases (p=0.025). Although there was no disease subtype uniquely associated with total or co-dominant multi-SF MT cases, occurrence of multiple SF mutations was more common in males (8% vs. 3% in females), an effect driven by ZRSR2 mutations. Hemoglobin and WBC were lower in multi-SF MT cases, with the lower hemoglobin strongly driven by the codominant multi-SF MT cases (median 8.3 g/dL vs 9.4 g/dL, p=0.00046). Conclusions: These findings suggest that SF mutation co-occurrence in myeloid neoplasms is more prevalent than previously estimated (~7%). Furthermore, we identified 6 individuals harboring > 2 SF mutations, which has not been previously described. We validate specific SF variant pairs that appear to exist in the same clone and are working to confirm the clonal architecture and characterize global splicing aberration in selected cases with single-cell studies to fully uncover the mechanism by which negative epistasis is overcome.

Abstract Background: Germline predisposition states (GPS), including inherited bone marrow failure syndromes (IBMFS) are seen in 5-10% of individuals with myeloid neoplasms (MN). Given overlap with somatic driver genes, blood, saliva, and buccal swabs are suboptimal specimens for GPS testing in MN; the latter two due to contamination with blood cells(Jerez, 2024). Skin biopsy with fibroblast culture-derived DNA remains the gold standard, but the process is expensive, takes 6-8 weeks to complete, and fibroblast culture failure can occur in some patients with telomere biology disorders. Given the need to rapidly screen donors for HSCT and adjust therapy/conditioning in certain IBMFS, we developed a rapid hematologic germline testing algorithm using hair follicle-derived DNA. Methods The study was conducted after IRB approval through the Mayo Clinic GPS/IBMFS clinic. To perform PCR-based hair follicle testing, we obtained 15-25 hair follicles (bulbs) per patient. The bulbs were placed in a microcentrifuge tube and subjected to a 24-hour digestion and extraction protocol using a modified version of the QIAGEN DNA Purification from Dried Blood Spots protocol. DNA quality was assessed by Nanodrop and Qubit.Testing included either validation of known or suspected germline variants (e.g., RUNX1, DDX41, ETV6), or panel testing (e.g., GPS or IBMFS). Pathogenic variants were validated by repeat testing in a CAP/CLIA certified lab prior to medical decision making. 23 patients underwent whole exome sequencing (WES) in a CAP/CLIA lab, and 13 patients had concurrent germline testing using alternate DNA sources (8 skin biopsies, 1 saliva, 4 buccal swabs). Results Ninety-three patients (49 male, 53%) underwent hair follicle-derived germline DNA testing (median age 63 years). Indications for germline testing included the presence of a suspected variant on somatic testing with variant allele frequency (VAF) between 35%-60% ( n= 66, 71%), patient age <50 years (n=26, 28%), antecedent thrombocytopenia (n=15, 16%), presence of syndromic features (n=4, 4%), patient history of multiple malignancies (n=4, 4%), and positive family history (n=15, 16%). Time to reporting variants after receipt of hair follicle-derived DNA was <14 days. There were zero complications associated with the procedure. DNA quality as assessed by Nanodrop revealed a median nucleic acid concentration of 25.64 ng/μL. The median A260/A280 ratio was 1.86 (range 0.701-2.133) and the median A260/A230 ratio was 1.27 (range -8.193-9.223). DNA quality was insufficient for chromosomal microarray (CMA) assessments. Of 93 patients evaluated, 67 (72%) had ≥1 germline variant, with 3 having >1 germline variant for a total of 72 variants. Pathogenic/likely pathogenic variants identified included: DDX41 (30, 41.7%), TP53 (4, 5.6%), ANKRD26 (4, 5.6%), RUNX1 (4, 5.6%), GATA2 (3, 4.2%), TERT (3, 4.2%), JAK3 (2, 2.8%), SAMD9/SAMD9L (2, 2.8%), ATM (2, 2.8%), and 1 each (1.4%) in BCL2, BCOR, BPGM, CDKN2A, CEBPA, CHEK2, CSF3R, CXCR4, DNMT3A, ETV6, GATA1, MPL, MPO, POT1, PTPN11, RPL5, and SH2B3. In the 13 patients that had concurrent germline testing, results were identical in all cases, except for 1 false negative in a buccal specimen (PTPN11) and 1 case of germline mosaicism, where the TP53 variant was identified in the blood and hair follicle DNA but not in skin fibroblast DNA. After testing, 30 (44.7%) patients had a change in their management plan: 8 (27%) with DDX41-GPS were observed due to known latency of DDX41-mutant MN, 6 (20%) were referred for HSCT based on the germline variant (CEBPA, DDX41, GATA1, GATA2, RPL5, TP53), 6 (20%) were placed on an enhanced surveillance plan for cancer screening (1 ANKRD26, 3 TERT, 2 TP53), 4 (13%) patients undergoing HSCT had related donors removed from consideration as they were carriers (2 DDX41, 1 ANKRD26, and 1 SAMD9), 2 (7%) patients with neutropenia and underlying germline variants (1 ATM, 1 DDX41) had G-CSF therapy discontinued due to concerns for clonal stimulation, and 1 (3%) patient with a germline CDKN2A mutation (hematological diagnosis- CMML) was recommended to avoid radiation therapy for prostate cancer. Conclusion In summary, we validate a rapid and simple hair follicle-derived DNA based germline assessment for patients with hematological disorders and demonstrate a positive impact on clinical care in 44% of patients. We are currently optimizing DNA quality so that germline copy number alterations can be better detected using CMA.

Abstract Introduction: VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) is an aggressive auto-inflammatory disorder defined by somatic UBA1 mutations. UBA1 mutations arise in hematopoietic stem cells and bias differentiation toward myeloid lineages. Clonal involvement of monocytes and neutrophils in VEXAS is frequent. Recently, natural killer (NK) cells have been recognized within the UBA1 mutant clone; mutated NK cells have an exhausted phenotype and impaired cytotoxicity (Ganesan BioRxiv 2025; Breillat et al, Blood 2025). A unique NK-like T cell type (CD3+CD16+[CD56-] T cells) has a role in the immunopathogenesis of severe COVID-19, resulting in heightened cytotoxicity and tissue damage (Georg et all, Cell 2022). As NK cells do not typically express CD3, and as the activation of inflammatory pathways is not restricted to myeloid cells in VEXAS, (Mizumaki et al, Nat Comm 2025) we sought to assess the CD3+ cell populations for co-expression of CD16 using single-cell (sc) multiomic data from VEXAS patients. Methods: After IRB approval, we performed sc-proteogenomic sequencing using a custom-made gene panel (Tapestri, MissionBio) in 2 VEXAS patients. Libraries were sequenced on an Illumina NovaSeq instrument. In one case, we performed in parallel CITE-Seq in parallel and used canonical correlation analysis (CCA) to integrate these immunophenotypic and transcriptomic data with matching cell populations identified from proteogenomic sequencing. We used pathway analysis (IPA) and gene ontology enrichment (gProfiler), as well as differential gene expression (RNA-seq) to identify significant gene signatures. T-tests with Welch's correction were performed for statistical significance on surface markers from proteogenomic data which were transformed by center log ratio. Our observations were then validated in sc-proteogenomic data from two additional VEXAS patients profiled at the NIH. Results: We used a sc-multiomic strategy to study CD3+ T cell subsets in VEXAS patients. In the first UBA1 mutant (mt) sample, using clusters defined by UMAP overlay of CITE-Seq/proteogenomic data, we defined positive expression of CD3 expression in the non-UBA1mt T cell cluster. We then examined the proteogenomic data (n=700 cells) to identify CD3+ cells (n=89 UBA1mt, n=42 UBA1wt). We then divided the CD3+ fraction into CD56+ vs CD56- to obtain CD3+CD56+ (n=25 UBA1mt, n=12 UBA1wt) and CD3+CD56- (n=64 UBA1mt, n=0 UBA1wt) cells. The CD3+CD56- cells were not only entirely UBA1mt, but also co-expressed CD16, significantly elevated from CD3+CD56+ UBA1wt cells (p<0.0001; and no significance from granulocyte populations): 10% of UBA1mt cells were CD3+CD16+CD56-. This cell subset was detected in an additional 3 VEXAS samples, and, in each case, there was no significant difference between UBA1mt/UBA1wt CD3 expression (both were T cell subsets) and no significant difference in CD56 expression. There was a significant increase in CD16+ expression in UBA1mt over UBA1wt (p<0.0001) cells, but not between UBA1mt cells and other granulocytes. Finally, we assessed the CITEseq data from the first UBA1mt case and focussed on gene expression and pathway differences between CD3+ and CD3+CD16+(CD56-) cell populations. Using IPA, we found top differentially expressed pathways included neutrophil degranulation, inflammation, cell killing, and IL-8 activity. Differentially expressed genes included FCGR3B, TLR2, IL1R2, FCGR2A, CCR1, CXCL8, and CXCR1, some of these overlapping with what was reported in similar cells (CD3+CD16+) detected in COVID-19 (Georg et all, Cell 2022). Conclusion: We identify a unique NK-T cell like [CD3+CD16+(CD56] subset of cells within UBA1mt cells in patients with VEXAS. This cell type has been reported in patients with severe COVID-19 as having increased cytotoxic functions and associated with fatal outcomes. Rare or aberrant cell clusters defined by standard clustering analysis may mis-identify some cell types, especially if they aberrantly express CD3+CD16+, as either marker could be used to pre-gate cells into larger clusters of lymphoid or myeloid cells. Prospective and functional profiling of this cell type in VEXAS patients is ongoing.

Abstract Background: DNMT3A and TET2 are epigenetic regulator genes commonly mutated in age-related clonal hematopoiesis (CH). Despite having opposed epigenetic functions, these mutations are associated with increased all-cause mortality and a low risk for hematological neoplasms. In this study, we make use of a natural inflammatory response occurring during COVID-19 to define the impact of these mutations on inflammation and outcomes. Methods: A cohort of 243 community-based patients (Olmsted County, MN) with COVID-19 were identified. CH status was determined on PBMC using error corrected sequencing (lower detection limit 0.5% VAF). In a subset of patients, we use multi-omics interrogation of the genome, transcriptome, and epigenome at single cell (sc) resolution along with DNA methylation, in PBMC, from patients with COVID-19 and CH. Results: We detected 97 CH mutations in 72 (29.6%) of 243 patients with COVID-19. The most frequent being DNMT3A (n=30, 30%) and TET2 (n=26, 28%). The presence of CH negatively impacted overall survival when restricted to TET2 and DNMT3A mutations. However, only DNMT3Amt CH retained age and comorbidity independent prognostication (HR 2.84, p = 0.022). We then assessed for changes in DNA methylation using Illumina MethylationEPIC array on patients with COVID-19 and DNMT3A (n=3) or TET2mt (n=4) CH. Site specific DNA methylation changes revealed significant loss of methylation in DNMT3Amt CH in comparison to TET2mt patients, with 10,944 hypomethylated and 1,160 hypermethylated sites. We found that actively transcribed states (Tx, TxWk) were more commonly hypomethylated in DNMT3Amt CH while enhancers (Enh) and promoters (TssA, TssAFlnk) were more commonly hypomethylated in TET2mt CH. We then conducted scDNA-seq in DNMT3Amt (n=3) and TET2mt (n=1) CH in COVID-19 and observed that while in TET2mt CH, mutations were largely restricted to classical and intermediate monocytes, in DNMT3Amt CH, mutations were seen in myeloid and lymphoid lineage cells. We performed scRNA-seq on patients with COVID-19 and DNMT3A (n=3) or TET2mt (n=6) CH. Differential gene expression analysis identified 1,569 upregulated genes in DNMT3Amt patients and 205 downregulated. Pathway analysis demonstrated an upregulation of genes involved in T cell survival, function, and inflammation in DNMT3Amt patients compared to TET2mt patients with a significant overexpression of GIMAP1, GIMAP4, IRF2, IL32 in multiple cell types. We correlated transcriptomic changes with chromatin accessibility using the 10X Genomics Multiome platform to profile both gene expression and open chromatin from the same samples. Analysis of global distribution of cut sites and differentially accessible peaks showed increased chromatin accessibility in DNMT3Amt CH, especially CD4+ T lymphocytes and NK cells. Conclusion: We demonstrate an age, sex, and comorbidity-independent adverse impact on mortality associated with DNMT3Amt CH in the context of COVID-19 and highlight transcriptomic and epigenetic differences between DNMT3A and TET2mt CH, with single cell resolution. Citation Format: Jenna A Fernandez, Wazim Mohammed Ismail, Moritz Binder, Terra Lasho, Susan M. Geyer, Amelia Mazzone, Christy M. Finke, Abhishek Mangaonkar, Jeong-Heon Lee, Liguo Wang, Vernadette A. Simon, Fariborz Rakhshan Rohakthar, Amik Munankarmy, Jonathan J. Harrington, Melissa R. Snyder, Keith D. Robertson, Alexandre Gaspar-Maia, Mrinal Patnaik. Divergent impacts of DNMT3A and TET2 mutant clonal hematopoiesis on COVID-19 related outcomes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Methylation, Clonal Hematopoiesis, and Cancer; 2025 Feb 1-4; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2025;85(3 Suppl):Abstract nr A012.

Abstract Background: Age-related clonal hematopoiesis (ARCH) is a precursor state to blood cancers and is associated with epigenetic age acceleration (EpiAgeAccel). While ARCH is associated with increased all-cause mortality and cardiovascular disease, not much is known about these risks with therapy-related clonal hematopoiesis (t-CH) – CH occurring in the context of prior cytotoxic chemotherapy/radiation exposure. Methods: Patients were prospectively recruited for CH and EpiAge assessment from Mayo Clinic between 2022 and 2024. CH status was determined using error corrected sequencing on peripheral blood (PB) DNA (sensitivity 0.5% VAF). Patients with CH were classified as t-CH if they had received chemotherapy or radiation prior to CH detection. All other CH cases were classifed as ARCH. For EpiAge analysis, PB DNA was analyzed using the Illumina MethylationEPIC array. The resulting beta values were used to obtain epigentic age estimates using Horvath’s clock, Hannum’s Clock, PhenoAge, DunedinPACE, and estimated telomere lengths (DNAmTL). Results: 106 (62%) of 170 patients assessed were found to have CH. CH patients were classified into ARCH-63 (59%) and t-CH-43 (41%). While the median chronological age (69 vs 67 years) was similar between ARCH and t-CH, t-CH patients had lower Hgb [10.9g/dL vs. 13g/dL (p=0.0001)], lower platelet [146x109/L vs. 192x109/L (p=0.0132)] and lower WBC [4.4x109/L vs. 5.2x109/L (p=0.0345)]. The 3 most common ARCH mutations were TET2 (20%), DNMT3A (19%), and ASXL1 (14%), while PPM1D (27%), TET2 (18%), and DNMT3A (14%) were most frequent in t-CH. There was no significant difference in median EpiAge via Horvath (68 vs 67.5years p=0.2826), Hannum (65 vs 66 years p=0.9333), or PhenoAge (55 vs 56 years p=0.618) between ARCH and t-CH. However, DunedinPACE of aging was higher in t-CH (1.27 vs 1.18 bioyear/chronoyear p=0.0148) and median DNAmTL was shorter in t-CH (6.35 vs 6.49kb p=0.0247), compared to ARCH (Note patients with telomere biology disorders were excluded). Multivariable regression analysis adjusting for chronologic age, smoking status, and male sex showed that both ARCH and t-CH had EpiAgeAccel via Horvath(5.8years p=0.0001, 3.4 years p=0.051), Hannum(5years p=0.001, 4.5 years p=0.007), and PhenoAge (6.7years p=0.002, 7.6 years p=0.002) clocks. t-CH more than doubled DunedinPACE (0.2 bioyear/chronoyear, p<0.001) of aging compared to ARCH (0.08 bioyear/chronoyear, p=0.026). t-CH also demonstrated a significant decrease in DNAmTL (0.34kb, p<0.001) in comparison to ARCH (0.17kb, p=0.015). With a median follow-up of 20 months, t-CH was associated with worse overall survival in comparison to ARCH, and non-CH patients (p=0.013, 93% vs. 90% alive at 10 months, 87 vs. 72% alive at 20 months). Conclusion: Our prospective CH cohort study validates the association between CH and EpiAgeAccel using several different methylation clocks and for the first time highlights an increased pace of aging and shorter DNAmTL in the context of inferior survival outcomes in t-CH. Citation Format: Marissa Li, Jenna Fernandez, Kristina Kirschner, Tamir Chandra, Kristen McCullough, Sarah Aug, Terra Lasho, Christy Finke, James Foran, Cecilia Arana Yi, Yael Kusne, Talha Badar, Abhishek Mangaonkar, Naseema Gangat, Rachel Simon, Alejandro Ferrer, Mrinal Patnaik. A prospective cohort study of biological aging and epigenetic age acceleration in 106 patients with age and therapy-related clonal hematopoiesis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Methylation, Clonal Hematopoiesis, and Cancer; 2025 Feb 1-4; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2025;85(3 Suppl):Abstract nr A015.

ASXL transcriptional regulator 1 (ASXL1) is one of the three most frequently mutated genes in age-related clonal hematopoiesis (CH), alongside DNA methyltransferase 3 alpha (DNMT3A) and Tet methylcytosine dioxygenase 2 ( TET2) . CH can progress to myeloid malignancies including chronic monomyelocytic leukemia (CMML) and is also strongly associated with inflammatory cardiovascular disease and all-cause mortality in humans. DNMT3A and TET2 regulate DNA methylation and demethylation pathways, respectively, and loss-of-function mutations in these genes reduce DNA methylation in heterochromatin, allowing derepression of silenced elements in heterochromatin. In contrast, the mechanisms that connect mutant ASXL1 and CH are not yet fully understood. CH/CMML-associated ASXL1 mutations encode C-terminally truncated proteins that enhance the deubiquitinase activity of the ASXL-BAP1 “PR-DUB” deubiquitinase complex, which removes monoubiquitin from H2AK119Ub. Here, we show that ASXL1 mutant proteins interact with the euchromatic histone lysine methyltransferases 1 and 2 (EHMT1–EHMT2) complex, which generates H3K9me1 and me2, the latter a repressive modification in constitutive heterochromatin. Compared to cells from age-matched wild-type mice, we found that expanded myeloid cells from old (≥18-mo-old) Asxl1tm/+ mice, a heterozygous knock-in mouse model of CH, display genome-wide decreases of H3K9me2, H3K9me3, and H2AK119Ub as well as an associated increase in expression of transposable elements (TEs) and satellite repeats. Increased TE expression was also observed in monocytes from ASXL1 -mutant CMML patients compared to monocytes from healthy controls. Our data suggest that mutant ASXL1 proteins compromise the integrity of both constitutive and facultative heterochromatin in an age-dependent manner by reducing the levels of H3K9me2/3 and H2AK119Ub. This increase in TE expression correlated with increased expression of nearby genes, including many interferon-inducible (inflammation-associated) genes (ISGs).

Introduction: Telomere biology disorders (TBD) are multisystemic accelerated aging syndromes due to abnormal telomere length (TL) shortening and increased cancer risk. Pediatric-onset TBD is characterized by severely shortened TL and specific associations with germline pathogenic variants mainly in DKC1, TINF2, PARN genes and characteristic clinical features [bone marrow failure (BMF), abnormal nail/skin/oral mucosa]. Adult-onset TBD is genetically distinct (predominantly, TERT, TERC, and RTEL1 mutations) with varied organ involvement [interstitial lung disease (ILD), BMF, cirrhosis, or bone disease] and heterogenous outcomes. In our previous study of predominantly adult patients with short telomeres, multiple organ involvement (Mangaonkar A et al. Blood Cancer J 2021) predicted poor overall survival (OS). We sought to validate these findings in a larger, well annotated adult cohort of TBD patients. Methods: Patients with suspected TBD underwent comprehensive clinical and molecular evaluation through our TBD-dedicated clinic after regulatory approval. TL was assessed through clinical flow cytometry fluorescence in-situ hybridization (FlowFISH).Clinical and laboratory features, including pulmonary imaging, lung volumes/function testing, hepatic imaging (MR elastography/fibroscan) and bone density assessments were retrospectively abstracted. Germline and somatic genetic testing was performed on peripheral blood through targeted panels for known TBD and myeloid genes respectively. Cox proportional hazard model was used to identify significant variables that also retained independent prognostic impact. Results: Overall, 122 patients met criteria for TBD as defined by lymphocyte TL (LTL) <=10th percentile and/or a presence of a germline-TBD variant; 34 patients LTL <1st percentile, 84 1st and the 10th percentile (27 with TBD-germline variants) and 4 with only TBD-germline variants. Median age at diagnosis was 56 (18-77) years, with 72 (59%) males. Clinical features included presence of ILD (n=81, 66%), BMF (n=39, 32%), cirrhosis/hepatic fibrosis (n=19, 38%), bone disease osteopenia/osteoporosis/fractures, n=53, 70%), MDS/AML (n=11, 9%), non-hematological cancer (n=38, 31%), and head and neck cancer (n=2). Germline pathogenic variants were detected in 22 (18%), most frequently in TERT (n=10), RTEL1 (n=7) and TERC (n=2), while variants of uncertain significance (VUS) were seen in 35 (29%) patients, especially in TERT (n=20), TERC/RTEL1(n=6), NAF1 (n=4). Somatic variants in hematopoietic cells were detected in 37 (30%) patients with frequent alterations seen in DNA damage response and repair (PPM1D/TP53, n=11), and splicing component (U2AF1/SF3B1/ZRSR2, n=9) genes. At last follow-up [median of 3 (0-18) years], there were 26 (21%) deaths (most commonly, progressive ILD-18, cancer of lung/breast/pancreas-3, transplant-related complications-2). After censoring patients at the time of any transplantation (lung transplant-26, liver transplant-5, bone marrow transplant-8), the Kaplan-Meier estimate of median OS was not reached. Univariate survival analysis found adverse OS predicted by older age at diagnosis (P=0.006), presence of ILD (P=0.0002), hepatic fibrosis/cirrhosis (P=0.02), and somatic DNA repair variants (P=0.03). Multivariate model analysis found that only presence of ILD retained independent prognostic impact (HR=2, P=0.02). Among radiographic ILD subtypes, UIP was the most common (51%), followed by NSIP (10%), and others (6%), with median FEV1 of 2L (range: 0.5-4.6), FVC 2.4L (0.7-6.2), and TLC 4L (1.9-7.4). TBD-ILD patients were older (P<0.0001) with a male preponderance (P=0.03), 52 (64%) with multi-organ involvement, and had a less severe hematological phenotype [lower incidence of BMF (P<0.0001) and fetal hemoglobin elevation (P=0.01)], more frequent bone disease (P=0.0009), relatively longer telomeres in lymphocytes (P=0.04), and no particular association with any germline or somatic genetic alterations. Conclusions: Presence of ILD is an independent and significant predictor of adverse survival in adult TBD. Within adult TBD, patients with ILD represent a unique clinical subtype without any obvious genetic or pathologic correlates. Focusing on definitive therapeutic measures in this subgroup of adult TBD has the best chance to alter natural history and improve outcomes.

Introduction: VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) is an adult-onset autoinflammatory disorder defined by somatic UBA1 mutations in hematopoietic cells. VEXAS is associated with a high frequency of clonal hematopoiesis (CH- 60%); with the clonal landscape being dominated by DNMT3A mutations. In our prior study (Gutierrez-Rodrigues Blood 2023), we showed that DNMT3A mutations in VEXAS associated with increased mortality, but without a higher risk of MDS/AML. As DNMT3A is a critical epigenetic regulator, we conducted this study to define the epigenetic deregulation and impact on mortality of DNMT3Amt (mutant) CH in VEXAS. Methods: After IRB approval, 88 VEXAS patients from the Mayo Clinic and the NIH were retrospectively assessed, including 81 screened for CH by error-corrected DNA sequencing (ECS). DNA methylation changes were identified using the Illumina Infinium MethylationEPIC BeadChip on peripheral blood samples (PB). Cellular indexing of transcriptomes and epitopes (CITE)-seq was performed using the 10x Chromium 3' GEX platform; 16,600 pooled cells were subjected to UMAP dimensionality reduction, followed by Azimuth based cell identification. Genotyping of targeted loci with chromatin accessibility (GoTChA) was conducted on two PB DNMT3Amt samples from CCUS individuals (Izzo et al Nature 2024). Results: Patients' median age was 70 years (range: 56 - 89); 95% were white. Twelve out of 81 patients screened by ECS were found with a DNMT3Amt (15%), with a median variant allele fraction (VAF) of 42% (range: 6-46%). In this cohort, while the presence of any CH mutation did not impact survival, DNMT3Amt CH had an age-independent negative impact on mortality (5.5 years vs 8.7 years, p =0.005), without an increase in risk for MDS/AML. Changes in DNA methylation were profiled in 19 and 9 VEXAS patients without CH (Median Age: 68) or with DNMT3Amt CH (Median Age: 74), respectively. VEXAS samples with DNMT3Amt CH had >4,000 differentially methylated CpGs with 95% of the CpGs being hypomethylated compared to those without CH. Using the Roadmap Epigenomics project, we found that this hypomethylation was enriched at enhancer (Enh) sites and actively transcribed states (TxWk). Pathway analysis revealed that the hypomethylated sites were in or near genes involved in the development and differentiation of leukocytes, with IL-13 and IL-15 being identified as upstream regulators of the methylation changes. Bulk RNA-seq performed on PB of 2 and 3 VEXAS patients with and without DNMT3Amt, respectively, identified >1,500 differentially expressed genes in DNMT3Amt samples, 78% of these being overexpressed. Pathway analysis was enriched in gene pathways involved in the development of leukocytes, with IL-13 and IL-15 once again being identified as upstream regulators. Integration of methylation and transcriptomic data identified upregulation of several genes within the IL-13 and IL-15 pathways, with these regions correlating with a decrease in methylation. To assess if the epigenetic dysregulation is linked to DNMT3A mutations, we performed GoTChA-seq on two DNMT3Amt clonal cytopenia samples (CCUS). The comparison of the number of cutsites in DNMT3A wild type vs mt cells from the same individual demonstrated increased open chromatin within the IL-13 and IL-15 pathways in mt cells, both globally and in a genomic locus specific manner. CITE-seq performed on 3 VEXAS samples (2 DNMT3Amt CH vs 1 no CH) further showed that expression of genes involved in IL-13 and IL-15 pathway is significantly increased in CD4+ and CD8+ T cells from DNMT3Amt samples, suggesting a paracrine regulatory effect of T lymphocytes (mostly UBA1 and DNMT3A wild-type) in VEXAS. Pathway analysis of DEGs in these cells also revealed an enrichment of genes involved in inflammation and lymphocyte proliferation. Conclusions: Our study validates the age-independent, negative prognostic impact of DNMT3Amt CH on survival in VEXAS syndrome. DNMT3Amt mediates epigenetic and transcriptional priming associated with upregulation of IL-13/15 pathway in CD4/CD8 T cells, which are mostly DNMT3A and UBA1 wild-type, suggesting that the contribution of T lymphocytes to inflammation may be secondary to CH in the myeloid compartment.

Introduction: ASXL1-mutant Chronic Myelomonocytic Leukemia (CMML) is a high-risk myeloid neoplasm characterized by epigenetic remodeling and overexpression of leukemogenic driver genes. DLK1 (delta-like homolog 1) is a non-canonical Notch ligand, crucial for the proliferation of early hematopoietic progenitor cells and its expression is controlled by maternal imprinting. DLK1 expression is restricted to select progenitor populations, but was found to be overexpressed in several malignancies including ASXL1-mutant CMML. Here we investigate the molecular mechanisms that drive the aberrant re-expression of DLK1 and postulate potential targeted therapeutic interventions. Methods: We performed bulk RNA- and ChIP-seq (H3K4me1/3, H3K27ac/me3, H2AK119ub, H2BK20ac) on bone marrow mononuclear cells from 20 patients with CMML (11 ASXL1-mutant, 9 -wildtype). All libraries were sequenced on an Illumina NovaSeq instrument. Differential gene expression and histone modification occupancy between ASXL1-mutant and -wildtype patients were assessed. Single-cell RNA-seq was performed for a subset of patients to investigate the cell populations of interest for DLK1 expression. To assess DLK1 allelic expression, we identified a single nucleotide polymorphism (SNP) in the coding region of DLK1 (rs1802710) and used cases which were confirmed heterozygous by Sanger sequencing. DLK1 transcript levels were quantified by RT-PCR, and the relative abundance of alleles was measured using digital droplet PCR. We designed primers to amplify the region around our SNP of interest and utilized high mismatch discrimination locked nucleic acid (LNA) probes labeled with FAM (reference SNP allele) and HEX (alternate SNP allele) to quantify specific alleles. The number of RNA molecules originating from each allele was quantified by counting the number of droplets that fluoresced with the corresponding probe colors. Illumina 850K EPIC microarrays were used to evaluate DNA methylation. Drug studies were carried out in progenitor colony forming assays derived from ASXL1-mutant patients (bone marrow mononuclear cells) with the dual BRD4 and p300 / CBP inhibitor EP31670 (62 nM, 24-hour exposure), quantifying DLK1 expression by real-time RT-PCR. Results: This study included 20 patients with CMML (median age at diagnosis 74 years). All ASXL1 mutations were typical hotspot mutations (most commonly Gly646Trpfs*12) and the variant allele frequencies were compatible with heterozygosity (median 0.41). The spectrum of co-mutations was typical and included TET2 (23%), SRSF2 (16%), CBL (7%), among others. DLK1 was strongly overexpressed in ASXL1-mutant patients (log2 fold-change +4.0, FDR < 0.001). Allele-specific gene expression studies (n = 3) revealed the re-expression of the imprinted maternal DLK1 allele in ASXL1-mutant patients. Single-cell gene expression studies (n = 5) demonstrated that the DLK1 overexpression is restricted to the hematopoietic progenitor cell population. Differential methylation analysis between patients with ASXL1-mutant clonal hematopoiesis (n = 4) and matched controls (n = 8) identified two hypermethylated CpG islands in the DLK1 promoter region (cg18122767, Δß = +8.6%, p = 0.021 and cg15831296, Δß = +5.8%, p = 0.008). Histone modification analysis identified DLK1 as a p300 target gene by mapping H2BK20ac onto established ASXL1-mutant-specific enhancer regions (H3K4me1 + H3K27ac co-occupancy). The distal enhancer EH38D2760182 was predicted to bind p300 and to govern the expression of DLK1. ChIP-seq experiment in K-562 and Kasumi-1 cells showed the binding of BRD4 and p300 to EH38D2760182, respectively. Targeting BRD4 and p300 using EP31670 led to the downregulation of DLK1 expression (mean fold-change = 0.569, p < 0.001, n = 3) in progenitor colony forming assays. Conclusions: We observed the aberrant re-expression of the maternally imprinted DLK1 allele in patients with ASXL1-mutant CMML. Both DLK1 promoter hypermethylation and the genotype-specific activation of enhancers by p300 were identified as putative regulatory mechanisms. The distal enhancer EH38D2760182 was found to bind BRD4 and p300 and the pharmacologic disruption of BRD4 / p300 activity in model systems led to the downregulation of DLK1. The clinical-grade dual inhibitor of BRD and CBP / p300 EP31670 is currently being investigated in patients with ASXL1-mutant chronic myeloid neoplasms (NCT05488548).