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Germline variants in DDX41 are the most common cause of inherited predisposition to Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML). DDX41 codes for an RNA helicase with many described functions, and thus the precise role of these variants in disease is uncertain. Approximately 2/3 of disease-associated DDX41 variants cause truncation of the DDX41 protein due to frameshift, loss of a translation start, or splice alterations, while the other 1/3 of DDX41 variants are missense. The effect of these missense variants on DDX41 function is largely unknown and thus they are typically categorized as variants of unknown significance (VUSs). Importantly, 30-50% of MDS/AML patients with either truncating or missense DDX41 variants acquire a somatic mutation in the other allele of DDX41, and this mutation has strong preference for the amino acid substitution R525H. Our previous work demonstrated that the R525H mutation causes loss of DDX41 function. To better understand how germline variants affect DDX41 function, we designed a genetic complementation strategy in immortalized hematopoietic progenitor cells with inducible deletion of DDX41. We screened six of the most common missense VUSs by expressing them from a lentivirus in DDX41-proficient cells and then inducing deletion of endogenous DDX41 to determine the effect of each VUS on proliferation and survival of the cells. Empty vector-transduced DDX41-deleted cells undergo cell-cycle arrest and apoptosis within 3-4 days. In contrast, viral expression of wild-type DDX41 rescues the survival and proliferation of the cells, while expression of the R525H mutant fails to rescue the cells. We screened the Y33H, M155H, G173R, R219H, P258L, and I396T variants of DDX41 in this assay, and all six of the mutants successfully rescued the survival and proliferation of DDX41-deleted cells long-term. This indicates that the mutant proteins coded for by these variants are functional. To test the effect of commonly observed truncating variants in the same assay, we expressed M1I, Q52fs, and D140Gfs variants of DDX41. Surprisingly, we saw complete rescue of cell proliferation and survival by the M1I and Q52fs variants. In contrast, the D140Gfs variant failed to rescue the cells. These data indicate that frameshift at D140 or later causes loss of DDX41 function. It is likely that translation from alternative methionine residues located at M127 or M132 of DDX41 accounts for the rescue of the cells by truncating variants affecting sites upstream of M127. In support of this theory, we targeted DDX41 for CRISPR-mediated knockout in MOLM13 cells and found that guide RNAs targeting exons 1-4, which are upstream of M127, produced homozygous frameshift mutations in proliferating cells from single-cell clones. However, guides targeting exon 6, which is downstream of M127 and M132, did not produce any surviving cells with homozygous frameshift mutations, despite screening 96 clones. This finding indicates that short DDX41 isoforms expressed from alternative start sites are functional and can support cell proliferation when full-length protein is lost. Collectively, these data raise important questions about the precise role of DDX41 variants in hematologic malignancy. Data from recently published patient cohorts indicates that patients with missense DDX41 variants have similar patient characteristics to those with truncating variants and thus these aberrations are thought to cause disease by similar mechanisms. While these cell line models may not fully recapitulate the pathogenic mechanism by which DDX41 variants cause MDS/AML predisposition, the results of this study demonstrate that the germline variants are not complete loss-of-function alleles in all cases and more complex mechanisms may be involved.

Introduction The advent of venetoclax (VEN) based chemotherapy has revolutionized the treatment of elderly acute myeloid leukemia (AML), with improvement in remission rates from historical 10%-20% to 60%-70%. However, remission duration is short-lived in patients (pts) with high-risk AML with consequent dismal outcome. As our knowledge of genomic landscape of AML expands and more immunotherapies become available, exploration of the immune tumor microenvironment (TME) of AML may allow identification of subsets of pts that may benefit from immune based therapy. We conducted an investigator initiated prospective study to evaluate TME of pts receiving VEN plus hypomethylating agent (HMA; azacitidine or decitabine) combination. We hypothesize that these pts will cluster into immune-infiltrated vs. immune-depleted sub-types by high-definition immune cell profiling. Methods In 26 newly diagnosed AML pts, who were planned to receive VEN plus HMA therapy, we isolated bone marrow (BM) and peripheral blood (PB) derived mononuclear cells (MC) by density gradient centrifugation and cryopreserved. We performed CyTOF (cytometry time-of-flight) analysis to study their immune signatures. Thawed cells were labeled with metal-conjugated antibodies that bind CD33, CD196, CD19, CD127, CD11b, CD4, IgD, CD11c, CD14, CD56, CD123, TCRgd, CD185/CXCR5, CD45RA, CD27, CD28, CD66b, CD183 (CXCR3), CD161, CD45RO, CD197 (CCR7), CD8a, CD25, CD3, CD20, CD38, HLA-DR, CD194 (CCR4), CD57, CD16 and CD45. We performed a Rphenograph clustering on 25 paired BMMC and PBMC samples (one patient only had PBMC) to create 35 unique clusters for each ( Figure 1). Then we broke down the distribution of cluster by two different groupings: pts with TP53 mutation (m) and those who achieve complete remission. Statistical significance of clusters calculated using t-test. We performed Pearson correlation test on the paired BMMC and PBMC samples to assess correlation between BMMC and PBMC samples. Results Baseline characteristics The median age of the pts was 74 years (range, 53-88), and 17 (65%) pts were male. Ten (37%) pts had secondary AML, and 12 (46%) pts had ELN adverse risk disease (n=6; TP53m). There was no significant difference in median age (p= >0.99), proportion of pts with secondary AML (p= 0.64), acute myelomonocytic leukemia (p= 0.14) white blood cell count (WBC) ≥ 100 (10 9/L) (p= >0.99), and frequency of commonly occurring co-mutations ( ASXL1 [p=0.35]), FLT3 ITD [p=0.54], splicing [p=0.29], IDH1/2 [p=0.28], DNMT3A [p=0.29], NPM1 [p=0.29], TET2 [p=0.35]) in TP53m and TP53 wild-type group. Response 12/20 (60%) evaluable pts achieved complete remission with or without count recovery (CR/CRi). There was no significant difference in median age (p= 0.06), proportion of pts with secondary AML (p= 0.16), therapy related AML (p= 0.60) WBC ≥ 100 (10 9/L) (p= 0.40), and frequency of commonly occurring mutations ( TP53 [p=0.60], ASXL1 [p > 0.99]), FLT3 ITD [p=0.54], splicing [p > 0.99], IDH1/2 [p=0.60], DNMT3A [p=>0.99], NPM1 [p=0.61], TET2 [p >0.99]) in CR/CRi and no-CR/CRi group. CyTOF analysis and Rphenograph clustering on 25 paired BMMC and PBMC samples We found significant clustering with high expression of B-cell activated (cluster 2; p= 0.005), NK cell rich (cluster 1; p= 0.005, cluster 35; p= 0.0009), CD8 T-cells (cluster 31; p= 0.01) and CD4 effector cells (cluster 17; p= 0.005) phenotype among TP53m AML ( Table 1). Among pts who achieved CR/CRi, significant clustering with low expression of B-cell activated (cluster 29; p= 0.01), CD8 cytotoxic (cluster 14; p= 0.01) CD4 activated (cluster 21; p= 0.03) phenotypes observed. Correlation between BMMC and PBMC clustering Among clusters which showed significance for a particular phenotype, we performed Pearson correlation (R 2) and observed strong correlation between BMMC and PBMC samples clustering in cluster 1 (R 2 = 0.99), cluster 2 (R 2 = 0.95), cluster 17 (R 2 = 0.96), cluster 21 (R 2 = 0.99) and cluster 29 (R 2 = 0.93). Conclusion In this single institutional study using CyTOF analysis we have identified novel clustering with immune- infiltrated phenotype among pts with TP53m AML and conversely immune-depleted phenotype among AML pts who responded to VEN plus HMA. Moreover, we found strong correlation in CyTOF analysis conducted from BMMC and PBMC samples. Future studies with larger sample size needed to validate our findings.

Background and Significance: The rare nature and diagnostic difficulty associated with myelodysplastic/myeloproliferative neoplasms (MDS/MPN) has limited the inclusion of patients (pts) with these diseases, other than chronic myelomonocytic leukemia (CMML), in relevant clinical trials. This has resulted in the complete lack of approved agents for the treatment of pts with non-CMML MDS/MPN. Moreover, ASTX727 (oral decitabine-cedazuridine) was approved for CMML based on a study of only 16 pts with CMML. A larger study of CMML and non-CMML MDS/MPN is needed. MDS/MPNs appear to be BCL2-dependent (Bogenberger et al., 2015; Jilg et al., 2016), but data for venetoclax combination therapy are derived from retrospective analyses of relapsed/refractory disease (Cortes et al. 2019; Hammond et al., 2020) or single-arm studies not dedicated to MDS/MPN (NCT04655755, Venugopal et al. 2021). Venetoclax exposures are not affected by coadministration of ASTX727 and predict responses similar to historical data (Mannis et al. 2021). Based on these promising early data and the critical need, we proposed a randomized study of ASTX727 alone or in combination with venetoclax for pts with MDS/MPN and >5% blasts. Study Design and Methods: This is a National Cancer Institute-sponsored, multi-center, international, randomized phase 2 study (NCT05600894, NCI 10538) designed to evaluate the efficacy of ASTX727 alone or in combination with venetoclax in pts with newly diagnosed MDS/MPN with >5% marrow blasts. Key eligibility criteria include allowing hydroxyurea cytoreduction to a WBC <10,000/mm 3 for proliferative disease and excluding pts with need for emergent disease-directed therapy as well as prior therapy for myeloid malignancy other than hydroxyurea or thrombopoietic/erythropoietin stimulating agents. Pts will be randomized 1:1 to receive either ASTX727 alone (Days 1-5) or in combination with venetoclax, initially administered with a 3-day dosing ramp-up and given for 14 days per 28-day cycle (Figure 1). Pts are stratified by disease subtype, specifically CMML and non-CMML MDS/MPN. The primary endpoint is the rate of complete remission (adapted from the MDS/MPN-specific response criteria developed by Savona et al. 2015). Secondary endpoints include the overall response rate, overall survival, progression-free survival, allogeneic hematopoietic stem cell transplantation rate, clonality at time of hematologic remission, number of red cell and platelet transfusions required and toxicity of ASTX727 versus ASTX727 + venetoclax. Pts randomized to the ASTX727 monotherapy arm and without response after 4 cycles of therapy, or with progressive disease after an initial response, cross over to the ASTX727 + venetoclax arm. A futility analysis is planned for each cohort (first 18 and 13 pts in each treatment arm for the CMML and non-CMML cohorts, respectively). We assume that 85% and 90% of pts in the CMML and non-CMML MDS/MPN cohorts, respectively, randomized to the ASTX727 monotherapy arm will not achieve the primary endpoint and will cross over to the ASTX727 + venetoclax combination arm. The estimated accrual time of a total of 70 pts with CMML and 50 pts with non-CMML MDS/MPN will require about 2.9 and 2.1 years, respectively. Planned integrated studies include the use of an apoptosis multiplex immunoassay to evaluate how the addition of a BCL-2 inhibitor will influence the ability of ASTX727 to induce apoptosis and if particular thresholds of apoptosis are predictive of clinical response. Planned exploratory studies include the use of: 1) RNA-seq to evaluate if differences in baseline transcriptional programs appear to be predictive of response to ASTX727 and ASTX727+venetoclax, 2) next generation (Oncomine Comprehensive Assay (OCAv3)/whole exome sequencing to uncover biomarkers predictive of response/outcome, validate risk modeling to stratify these pts and detect/monitor measurable residual disease (MRD) on serial marrow samples that can predict depth of response with translation in improvement in event-based endpoints, and 3) circulating tumor DNA as a non-invasive means (i.e., without need for a marrow evaluation) of prospective MRD detection for prognostic use. This ongoing, randomized study may inform frontline therapeutic options, provide prospective data for venetoclax combination after HMA failure, and generate striking correlative science for pts with “high risk” MDS/MPN.

Introduction: MDS are primarily diseases of the elderly, with median age at diagnosis of 72 years. Focused data on clinical and molecular characteristics, as well as outcomes, in younger pts under age 60 years are limited. We conducted this multicenter retrospective study to describe pt characteristics, cytogenetic and molecular profiles, treatment modalities, and outcomes in younger pts with MDS, including adolescent and young adults (AYA). Methods: The study included pts diagnosed with MDS between 2002-2023 at 7 academic centers. We compared baseline characteristics, treatment, and outcomes of AYA (18-39 years) and younger adult (YA) pts (40-59 years). Responses were assessed using the modified IWG 2006 response criteria which included complete remission (CR), marrow CR (mCR), and hematologic improvement (HI). Wilcoxon rank sum test and Fisher's exact (and Chi-square) test were used to compare pt and disease characteristics for continuous and categorical variables, respectively. Overall survival (OS) was estimated using the Kaplan-Meier method and compared using log-rank test. Results: A total of 173 pts were identified (AYA, N=50; YA, N=123). Median age at diagnosis was 50 years (range, 18-59), with 51% females. Baseline characteristics are shown in Table 1. Therapy-related MDS (t-MDS) was observed in 40 pts (23%) with the remaining classified as de novo MDS (N=133; 77%). Inherited bone marrow failure syndromes were documented in 15 pts (9%), with Shwachman-Diamond Syndrome (N=4) and Fanconi anemia (N=4) being the most frequent. Using the WHO 2016 classification, the most common subtypes of MDS in both the AYA and YA cohorts were MDS with multilineage dysplasia (38% vs 19%), MDS with excess blasts-2 (MDS-EB) (24% vs 29%), and MDS-EB-1 (16% vs 20%). Similarly, using the WHO 2022 classification, the most common subtypes were MDS with low blasts (50% vs 21%), MDS with increased blasts-2 (MDS-IB-2) (24% vs 24%), and MDS-IB-1 (16% vs 18%). Stratifying pts using the IPSS-R cytogenetic (CG) risk group, the most common CG risk category identified was good CG (34% vs 52%, p=0.012). Poor/very poor CG were present in 34% and 37% of the AYA and YA groups, respectively. STAG2 and NRAS mutations occurred more frequently in the AYA pts compared to the YA group, while the YA cohort was enriched for the following mutations: TP53, DNMT3A, SF3B1, SRSF2, and TET2 (Figure 1). In the AYA population, 18 pts (38%) had very low/low, 10 (21%) intermediate, and 20 pts (42%) had high/very high risk MDS using IPSS-R. In the YA population, 32 pts (26%) had very low/low, 32 (26%) intermediate, and 57 pts (47%) had high/very high risk MDS using IPSS-R. Twenty-seven percent of pts with very low to intermediate-risk MDS using IPSS-R were upstaged to moderate high to very high-risk MDS using IPSS-M. The majority of pts (AYA: 75%; YA: 63%, p=0.5) were transfusion independent at baseline. Single-agent hypomethylating agents were the most frequently used frontline therapy, used in 17 AYA pts (34%) and 58 YA pts (48%), with more AYA pts receiving upfront allogeneic stem cell transplantation (Allo-SCT) compared to YA pts (28% vs 5%). After frontline therapy (Table 1), an additional 14 AYA (28%) and 52 YA (42%) pts received Allo-SCT. In pts with IPSS-R intermediate to very high-risk, response rates (HI+CR+mCR+mCR with HI) to frontline therapies were 46% and 54.5% in the AYA and YA groups, respectively. After median follow-up of 24 months [range, 1-218], median OS was numerically longer in the AYA versus YA group (155 vs 53 months, p=0.2). AML transformation occurred in 24% and 20% of the pts in the AYA and YA groups, respectively. Median OS was significantly longer in de novo compared to t-MDS pts (56 vs 27 months, p=0.03). Median OS among Allo-SCT compared to non-SCT pts were not reached vs 155 months ( p=0.9) in the AYA group and 53 vs 54 months ( p=0.4) in the YA group. IPSS-R (very low+low vs intermediate vs high+very high) was able to distinguish differences in OS in the YA (133 vs 53 vs 17 months, p=0.0002) and AYA (155 vs not reached vs 18 months, p=0.006) pts, with the exception of the intermediate-risk group in the AYA cohort. Conclusions: In this study, d e novoMDS was seen in the majority of younger pts with MDS (both AYA and YA). Mutations in STAG2 and NRAS were more common in the AYA pts, while YA pts had MDS more enriched for TP53, DNMT3A, TET2 and splicing mutations. This study is still ongoing with a plan to compare the abovementioned groups to older pts (≥60 years) with MDS.

Introduction Chronic myelomonocytic leukemia (CMML) can be classified into dysplastic (dCMML) and proliferative (pCMML) subtypes based on a white blood cell count (WBC) ≥ 13 x 10 9/L for pCMML. Though this cutoff may seem arbitrary, there is evidence that pCMML is a unique biological entity with a distinct molecular profile and worse outcomes (Carr RM, et al. Nat Commun. 2021). We hypothesized that contemporary CMML prognostic scoring systems - such as the Mayo Molecular Model (MMM; Patnaik MM, et al. Leukemia. 2014) and the CPSS-Molecular Model (CMM; Elena C, et al. Blood. 2016) - would not adequately risk-stratify pCMML patients. Moreover, while truncating ASXL1 mutations are considered a high-risk feature in CMML, their prognostic relevance in pCMML is unknown. Methods After IRB approval, we compiled a large (n = 888), molecularly-annotated database of CMML patients seen at two US medical centers (Mayo Clinic and MD Anderson) via retrospective chart review. All statistical analyses considered the clinical and laboratory parameters obtained at the time of presentation. The cohort was divided into dCMML and pCMML subgroups, which were analyzed independently alongside the entire cohort. Categorical variables were compared by Fisher exact or Pearson χ 2 tests and continuous variables by Mann-Whitney U tests. Univariate and multivariate analyses were performed using Cox proportional hazards regression models. Survival was assessed via the Kaplan-Meier method. P-values < 0.05 were considered significant. Calculations were performed using the BlueSky Statistics (v10.3.1) interface for R. Results We identified 474 (54%) dCMML cases and 410 (46%) pCMML cases with median age 71 years and 67% males. Patients with pCMML were more likely to have higher AMC (5.1 vs 1.5 x 10 9/L, p < 0.0001), circulating immature myeloid cells (IMC; 65% vs 36%, p < 0.0001) and abnormal karyotypes (37% vs 30%, p = 0.0187). They were also overrepresented in higher-risk categories of the MMM and CMM compared to dCMML (p < 0.0001 for each model). The number of somatic mutations was higher in pCMML than dCMML (mean 3.0 vs 2.6, p = 0.0050). Mutations in ASXL1 (53% vs 39%), NRAS (21% vs 11%), CBL (18% vs 12%), SETBP1 (14% vs 7%), JAK2 (10% vs 3%), CEBPA (5% vs 2%), GATA2 (3% vs 0%), and FLT3 (3% vs 1%) were more frequent while mutations in TET2 (38% vs 48%), SF3B1 (2% vs 8%), ZRSR2 (2% vs 8%), and NPM1 (1% vs 3%) were less frequent in pCMML compared to dCMML, respectively (p < 0.05 for all). Though the MMM and CMM effectively stratified the dCMML group, they did not provide meaningful risk stratification in the pCMML group (MMM p < 0.05 overall but could not distinguish between low and intermediate-1/2 or intermediate-1 and high risks; CMM p > 0.05). Likewise, there was no difference in the median overall survival (OS) or leukemia free survival (LFS) when pCMML patients were stratified by truncating ASXL1 mutations( Figure 1A). We then sought to better risk stratify pCMML. Univariate analysis identified increasing age, male gender, abnormal karyotype, and elevated WBC, ANC, and AMC as adverse predictors for OS. In addition, the presence of IMC and peripheral blood (PB) blast % were adverse factors for LFS, while age was not significant for LFS. None of the aforementioned mutations were significant in the univariate model for OS, whereas GATA2 and NPM1 mutations were significant for LFS. In multivariate analysis for OS, only male gender and AMC ≥ 10 x 10 9/L retained significance. In multivariate analysis for LFS, male gender, AMC ≥ 10 x 10 9/L, and peripheral blasts ≥ 5% remained significant; GATA2 and NPM1 mutations were not significant. These variables risk-stratified pCMML patients based on the presence of 0, 1, or ≥ 2 risk factors ( Figure 1B). In comparison, OS was 40.7 months (95% CI 35.2 - 50.1 mo.) for dCMML patients, 20.1 months (95% CI 18.2 - 22.7 mo.) for pCMML patients, and 31.8 months (95% CI 28.4 - 33.5 mo.) for the entire cohort. At last follow up, 586 deaths (66%) and 168 AML transformations (19%) were recorded in the cohort. Deaths in dCMML vs pCMML were 294 (62%) vs 291 (71%; p = 0.0067), with AML transformations in 83 (18%) vs 84 (21%; p = 0.2055) patients, respectively. Conclusions Truncating ASXL1 mutations, along with ASXL1-mutation weighted CMML prognostic models failed to effectively risk stratify pCMML patients. In pCMML, male gender and AMC ≥ 10 x 10 9/L, effectively risk stratified for OS, while these factors along with PB blasts ≥ 5% effectively risk stratified for LFS.

BACKGROUND: Recent World Health Organization (WHO) classifications (5 th edition), International Consensus Classification (ICC) recognized TP53-mutated ( TP53mut) myeloid neoplasms (MNs) as a distinct subcategory, acknowledging their uniformly poor outcomes. However, there are critical differences between the two guidelines in how TP53 allelic status is assigned. Both WHO-5 and ICC acknowledged the poor outcome of multi-hit TP53mut but excluded single-hit TP53mut. These changes are driven by a pivotal study demonstrating favourable outcome of single-hit TP53mut, however this study predominantly included de novo myelodysplastic syndromes (MDS) cases (Bernard et al. Nat Med 2020). In contrast, we previously reported poor outcome of TP53mut therapy-related myeloid neoplasm (t-MN) irrespective of the allelic status of TP53 (Hiwase et al Blood 2022andShah et al BCJ 2023). Finally, the ICC removed the subcategory of “therapy-related,” substituting it with diagnostic qualifiers instead. The WHO has grouped t-MN with secondary MN and renamed it as “myeloid neoplasm post cytotoxic therapy,” with the assertion that only multi-hit T P53mut MDS occurring post-cytotoxic therapy have poor outcome compared with single-hit. METHODS: We analyzed one of the largest international cohorts of TP53mut MN (n=544) with full characterization of TP53mut allele status utilizing uniform ICC criteria and similar large numbers of de novo and t-MN cases managed at Mayo Clinic Enterprise (USA) and South Australian MDS Registry, Adelaide (Australia) with clinical, integrated genomic profiling, and long-term follow-up. RESULTS: In our cohort of TP53mut MN (n=544), 475 (87.3%) MN had TP53mut variant allele frequency (VAF) of ≥10% and thus satisfied ICC criteria for TP53mut MN. Majority of TP53mut t-MN presented with MDS (bone marrow (BM) blasts 0-9%) compared de novo MN (62% vs. 48%; P=0.0012). While acute myeloid leukemia (AML) (BM blasts ≥20%) were more prevalent in TP53mutde novo MN compared to t-MN (38% vs. 24%; P= 0.001) and there was no difference in frequency of MDS/AML (BM blasts 10-19%) between the two cohorts (14% vs. 14%; P=0.8) ( Figure 1A). Majority of TP53mut were observed in the DNA-binding domain in t-MN and de novo MN (95% vs. 96.2%; P=0.48). However, mutations in Tetramer Domain were more frequent in t-MN (4.3% vs. 0.3%; P=0.002), whereas those in the TAD2 domain were more frequent in de novo TP53mut MN (2.4 vs. 0%; P =0.006). Integrating the mutation, conventional G-banding cytogenetic, copy number variation (CNV), FISH, and SNP array analysis, TP53mut MDS (BM blast 0-9%; n=260) were classified as multi-hit (n=179, 68.9%) or multi-equivalent (n=35, 13.5%) and single-hit (n=46, 17.7%) (Arber et al. Blood 2022). Striking enrichment of multi-hit/multi-hit equivalent was observed in TP53mut t-MDS compared to de novo MDS (91% vs. 72%; P<0.0001). While single-hit TP53mut were prevalent in de novo MDS compared to t-MDS (28% vs. 9%; P<0.0001). Importantly, median overall survival (OS) of single-hit was significantly longer than multi-hit de novo MDS (25.4 vs. 7.2 months; P=0.0002). In contrast to this we did not observe OS difference between single-hit and multi-hit TP53mut t-MDS (11.6 vs. 10.9 months; P =0.73) ( Figure 1B). CONCLUSION: Our study based on a large international cohort of T53mut MN provides compelling evidence that in contrast to de novo MDS, single hit TP53mut t-MDS is associated with poor outcome. Our data supports the consideration of single hit t-MDS as a multi-hit equivalent.

Background: Clonal cytopenia(s) of undetermined significance ( CCUS) is defined as persistent cytopenias arising in the context of myeloid neoplasm ( MN)-associated somatic mutations ( MT) in hematopoietic stem cells. Patients ( PTs) with TET2MT CCUS have a high probability of progression to MN. To date, no FDA-approved therapies exist for CCUS, and PTs often have similar cytopenias/transfusion needs as those with myelodysplastic syndromes ( MDS). TET2 is an ascorbic acid (AA)-dependent dioxygenase that catalyzes the conversion of 5-methylcytosine ( 5mC) to 5-hydroxymethylcytosine ( 5hmC), providing rationale to study high-dose IV (HI)-AA in TET2MT CCUS. Here we report the final results of this pilot trial. Methods: This is an investigator-initiated, single-institutional, phase II trial assessing the safety and efficacy of HI-AA in PTs with TET2MT CCUS (NCT03418038). PTs≥ 18 years with ≥1 TET2MT with or without additional somatic MTs, without prior treatment, and with any of the following laboratory criteria: (1) hemoglobin ( Hb) ≤10g/dL, (2) absolute neutrophil count ( ANC) ≤1(10^9/L), (3) platelet count ( PLT)≤100 (10^9/L) were eligible. HI-AA (1g/kg, maximum 100g) was given 3 times weekly for 12 weeks. The primary endpoint was hematologic response rates determined by MDS IWG 2018 criteria at week 20. (Platzbecker Blood 2018) and reported as hematologic improvement ( HI)- erythropoietic ( HI-E), platelets ( HI-P), neutrophils ( HI-N). Secondary endpoints include safety and adverse events ( AEs), graded by NCI-CTCAE v4.03. Correlative studies include changes in TET2MT variant allele fraction ( VAF), in vitro colony formation and differentiation, global and sequence-specific DNA methylation/hydroxymethylation, and quantitative 5hmC estimated by IHC in bone marrow biopsy specimens. Results: Ten patients were enrolled, with a median age of 71.4 (range: 65, 79) years, 8 (80%) males. The median number of mutations was 3, with 9 (90%) having co-mutations. All PTs but one [-Y] had normal karyotype. Baseline Hb levels were normal in all but one who was red blood transfusion dependent. Five (50%) PTs were thrombocytopenic, and 5 (50%) PTs had ANC<1 (10^9/L). ( Table). HI-AA was well tolerated with the most common AEs being infusion-related polyuria (40%) and polydipsia (40%, 3 Grade 1, 1 Grade 2). One (10%) PT had constipation and headaches (both Grade 1), and 1 (10%) experienced dyspepsia. No treatment-related Grade 3 or 4 AEs' or deaths were reported. The median follow-up duration was 16.8 months (range: 9.4, 24.1). Overall, no PTs met the criteria for HI. There were no significant differences with regards to median Hb, PLT, and ANC values at baseline, week 20, and 1-year (median Hb: 13.3, 12.5, and 12.7 g/dL, p=0.93; PLT: 97 vs. 120 vs. 92.5X10^9/L, p>0.99, and ANC: 0.9 vs. 1.2 vs. 1.3X10^9/L, p=0.99). By CTCAE grading criteria for cytopenias, 3 (30%) PTs had an improvement in severity of cytopenias, while 2 had worsening. One (10%) PT had an improvement in PLT (baseline vs. week 20: 62 vs 192X10^9/L) but was morphologically deemed to meet criteria for CMML-1 at week 20. Four (40%) PTs met criteria for disease progression (PT_4, 7,8,10); CMML-1- 2 and MDS-2. The median time to progression was 5 months (95% CI: 4.9, unreached). Two PTs had mutational clonal evolution, with 1 (PT_8) acquiring a RUNX1 (VAF: 20%) and 1 (PT_9) acquiring 2 CBL mutations (both VAF:4%) at one-year follow-up. There were no significant differences regarding the clinical characteristics or the TET2 variants between PTs who had stable disease vs. progression. There were no significant differences in TET2 VAF at baseline, week 20, and 1-year (median: 39% vs. 43% vs. 42%, p=0.54). We profiled changes in 5-mC and 5-hmC (Ilumina EpicArray) in PTs pre- and post-IV AA treatment and did not observe global changes. However, we were able to highlight site-specific changes in differentially methylated regions, largely resulting in hypomethylation at enhancer sites and affecting actively transcribed states in PTs with an improvement in cytopenia(s) severity by CTACE criteria ( Figure). Conclusion: We report the final safety and efficacy analysis of HI-AA in PTs with TET2 MT CCUS. The trial data suggest that HI-AA is safe and well tolerated. Although there were no significant responses by IWG MDS criteria, alleviation in severity of cytopenias in a subset of PTs (n=3) along with correlative epigenetic changes in enhancer regions, needs further exploration.

Introduction: Chronic myelomonocytic leukemia (CMML) is a myeloid neoplasm with limited therapeutic options and dismal outcomes. Truncating mutations in the epigenetic modifier ASXL1 are common and lead to a proliferative disease phenotype, resistance to epigenetic therapies, and higher rates of AML transformation. ASXL1-mutant CMML is characterized by widespread epigenetic remodeling and up-regulation of key leukemogenic driver genes (e.g. HOXA9 and its co-factor MEIS1). There is a genotype-specific activation of enhancers in ASXL1-mutant CMML that is associated with up-regulation of these leukemogenic driver genes. Here, we test the hypothesis that disrupting the oncogenic interactions at these genotype-specific enhancers can be exploited for therapeutic benefit. Methods: We identified activated enhancers specific to ASXL1-mutant CMML as previously described ( Binder et al. Nat Commun 2022;13(1):1434). To disrupt the E2542605 enhancer, we created unique mapped single guide RNAs (confirmed by BLAT). We generated a ribonucleoprotein complex using the Alt-R CRISPR-Cas9 system (IDT Technologies, Coralville IA). We combined the RNP along with Electroporation Enhancer to 3x10^5 cells of the monocytic cell line U937 in 20uL strip-wells provided by the SF Cell Line 4D-Nucleofector X Kit (Lonza, Basel, Switzerland). To deliver the RNP, U937 cells were electroporated via listed cell line specific protocol by the Amaxa 4D-Nucleofector (Lonza). Forty-eight hours post-electroporation, cells were split and sequenced for E2542605 genomic structure disruption. In parallel, RNA was harvested, converted to cDNA and qPCR for HOXA9 and MEIS1 expression was performed using PrimeTime qPCR FAM labeled probe assays (IDT). Gene expression was determined using delta-delta Ct method. For assessment of selective sensitivity of ASXL1 / enhancer interactions, we performed progenitor colony forming assays using primary bone marrow mononuclear cells from CMML patients with or without ASXL1 mutations and healthy normal controls (n=2 in each category), exposed to increasing concentrations of the dual p300/CBP and BET bromodomain inhibitor EP31670. Briefly, we plated cells in methocellulose fortified with recombinant human cytokines supporting hematopoietic growth in duplicate, in a range of EP31670 concentrations. After 10 days, visible hematopoietic colonies were quantified and characterized. IC 50 values were generated using non-linear regression log(EP31670) vs. normalized response. Results: We computationally identified candidate transcriptional regulators enriched at the activated enhancers specific to ASXL1-mutant CMML, including BRD4 (FDR<2.57x10 -370), CBP (FDR=2.56x10 -217), and p300 (FDR=6.36x10 -93). We selected the ASXL1-mutant-specific enhancer E2542605 (predicted to drive HOXA9 expression), given the up-regulation of HOXA9 in ASXL1-mutant CMML (log 2-fold change +2.05, FDR=0.006, n=14). We disrupted E2542605 using two different CRISPR guides, leading to a PCR trace detection of a 14bp deletion and a 26bp indel in the E2542605 enhancer region, respectively. Both enhancer disruptions led to a down-regulation of its putative target gene HOXA9: Mean relative fold-change 0.36 (p=0.001, n=3) and 0.65 (p=0.046, n=3), respectively ( Fig. 1a). We then used a pharmacologic approach (EP31670) to target the candidate transcriptional regulators involved in the oncogenic interaction at these ASXL1-mutant-specific enhancers. We observed selective sensitivity to growth of CMML colonies (particularly ASXL1-mutant colonies) compared to ASXL1-wild type cases and normal bone marrow controls ( Fig. 1b). Conclusions: In CMML patients, truncating ASXL1 mutations are associated with the activation of genotype-specific enhancers and the recruitment of an extended repertoire of transcriptional regulators including BRD4, CBP, and p300. In a model system, the disruption of an enhancer predicted to govern the expression of the leukemogenic driver HOXA9, led to a significant down-regulation of HOXA9 expression. Pharmacologically disrupting the oncogenic interaction at such enhancers with EP31670 in primary patient samples led to significant therapeutic effects at nanomolar drug concentrations in a genotype-specific manner. Oncogenic interactions at activated enhancers are viable therapeutic targets in ASXL1-mutant CMML and warrant further clinical investigation.

INTRODUCTION Telomere biology disorders (TBDs) are multisystem diseases caused by accelerated telomere shortening that can present with bone marrow failure and pulmonary fibrosis, among other abnormalities. TBD diagnosis is considered when average blood telomere length (TL) measured by clinical flowFISH is <1 st percentile and/or a pathogenic variant in telomere maintenance genes (most commonly TERT, TERC, RTEL1) is identified. Pathogenic variants in genes coding for the telomerase protein ( TERT) and RNA component ( TERC) decrease telomerase activity. Similarly, variants in RTEL1 result in defective unwinding of the t-loop at telomere ends, increasing the levels of free t-circles in cells. Variants in these genes are often classified as variants of uncertain significance (VUSs by ACMG criteria) with additional efforts needed for variant curation. To address this, we describe results of functional testing performed in TBD patients with VUSs involving TERT, TERC, and RTEL1. METHODS TBD suspected patients were identified through the Pre-Myeloid Cancer and Bone Marrow Failure Clinic (Mayo Clinic) where they underwent clinical flowFISH and genetic testing for TBD related genes. Informed consent was obtained with approval from the Mayo Clinic Institutional Review Board. Viable peripheral blood and bone marrow samples were collected along with age matched healthy volunteers. Telomerase activity was measured in samples with TERT and TERC variants through telomeric repeat amplification protocol (TRAP) assays using the TRAPeze XL Telomerase Detection Kit (MilliporeSigma) following manufacturer instructions. HCT116 cells were used as positive controls while heat inactivated samples served as negative controls. Fluorescence was measured in a SpectraMax plate reader. Percentage of total product generated (TPG) relative to healthy age matched controls was calculated. T-circle detection assays were used on RTEL1 samples based on the protocol by Zhang et al. (2017). Final southern blotting was completed using the TeloTAGGG Telomere Length Assay Kit (Roche), and t-circle bands were visualized in a c600 GEL Imaging System (Azure Biosystems). RESULTS Ten patients (mean age = 53 years (27-71), 50% female) were included in our study. Patients 1 to 5 presented VUS in TERT (n=4) or TERC (n=1) and TL <1 st percentile in lymphocytes and/or granulocytes. Patient 6 carried a known pathogenic variant in RTEL1 and was included as a positive control. Patients 7 to 9 with RTEL1 variants presented TL close to the 10 th or between the 1 st-10 th percentile. No TL results were available for patient 10 but was tested due to the presence of a VUS in RTEL1 (Table 1). Similarly, while patient 7 carried a likely pathogenic variant, it was tested due to presenting lymphocyte TL between the 1 st-10 th percentile. TRAP results indicated decreased telomerase activity compared to age matched controls in all samples with TERT and TERC variants suggesting a pathogenic effect (52.%, 10.7%, 48.7%, 4.4% and 5.2% of TPG compared to age matched controls, respectively) (Table 1). On the other hand, t-circles were only seen in patient 6 (Figure 2). DISCUSSION We used TRAP and t-circle assays to explore the telomerase activity and presence of t-circles in patient samples with TERT/TERC and RTEL1 variants. In the case of TERT/TERC, we observed a reduction of telomerase activity in all samples. Together with blood TL below the 1 st percentile, this suggests that these variants are indeed deleterious (Table 1). However, since patient 4 presented two TERT VUSs, additional testing is needed to conclude how each variant impacts telomerase activity independently. In regard to RTEL1 variants, we observed t-circles in patient 6 as expected. No t-circles could be seen on all remaining samples, including patient 7 who carried a variant classified as likely pathogenic (Figure 1). This in-frame deletion has been described once in a TBD family (Cogan et al. 2015) but was not functionally tested. This result, together with the borderline TL seen in this patient, may suggest that some compensatory mechanism exists on this sample, or that the deleterious effect of the variant is not strong enough to be detected by our assay. Further studies are currently ongoing by our group to validate these results using a cell-based transfection disease model. In summary, we show that functional testing can be a helpful tool to determine pathogenicity of VUSs in TERT/TERC and RTEL1.

Background: Clonal cytopenia of undetermined significance (CCUS) is characterized by the presence of a clone with somatic mutations in genes associated with myeloid neoplasms (MN) or abnormal cytogenetics, along with unexplained and persistent cytopenias in the absence of significant marrow dysplasia. CCUS is considered a premalignant state with a risk of progression to MN. The clinical outcomes of patients (pts) with high risk CCUS (Weeks et al, NEJM Evidence 2023) may be similar that of some patients with myelodysplastic syndromes (MDS). This study aims to predict the outcomes of CCUS patients using the molecular international prognostic scoring system (IPSS-M) developed for MDS. Methods: A retrospective analysis was conducted in patients diagnosed with CCUS between 2015 and 2023, we included patients harboring pathogenic mutations (MUT) identified through next-generation sequencing (NGS) testing at two institutions: Mayo Clinic and the University of Texas Southwestern Medical Center, Dallas, TX, USA were included. Progression was considered present if patients with CCUS progressed into myeloid neoplasm at follow up. Statistical analysis was performed using JMP® 17.1.0 Software. IPSS-M risk scores were calculated via the IPSS-M Risk Calculator (mds-risk-model.com). Results: A total of 103 patients were included in the study. The median age was 72 years, the majority were male patients (73%), and the median bone marrow blast % at the time of NGS was 1%. Forty-five % had an isolated mutation in a single gene, and 55% had co-mutations in ≥1 gene. The median number of mutations was 2 (range 1 - 5), occurring across 43 different genes. The most common mutations were in TET2 (N=28; 27%), ASXL1 (N=21; 20%), SRSF2 (N=21; 20%), U2AF1 (N=18; 17%), DNMT3A (N=16; 16%), and TP53 (N=12; 12%). Additionally, 25% of patients had an abnormal karyotype. Hematologic and genetic characteristics are summarized in (Table 1A). The median IPSS-M score was -0.9 (-3 to +2.34) and the median IPSS- Revised (R) score was 2 (0 - 6). The majority of patients had low (L) IPSS-M risk category (53%), followed by moderate low (ML) (19%) and very low (VL) (17%). Per IPSS-R, the majority had low-risk category (47%), followed by VL (38%) and intermediate (14%). Overall, 26 (25%) pt died after a median follow-up of 34 months and 21% progressed to MN (MDS 14 & CMML 8), and 1 patient with MDS and 1 with CMML progressed to secondary AML (2%). The median overall survival (mOS) was not reached, with a 2-year estimated (YE) OS and leukemia-free survival (LFS) of 80%. Predictive Power: When utilizing the IPSS-M risk category ( L, M, H) * by combining the risk categories into L *(L+VL), M * (ML+MH), and H *(H+VH) to overcome the low sample size limitation, it significantly predicted OS (p < .0001) ( Figure 1A) and median MN-free survival (FS) (p < .0001) ( Figure 1B). The 2YE-OS rates by IPSS-M risk category were as follows: VL - 93%, L - 87%, ML - 75%, high (H) - 25%, very high (VH) - 0% and the 2YE-OS rates by IPSS-M ( L, M, H) are summarized in Table 1B. IPSS-M hazard ratio (HR) for OS was 1.6 for ( M*) compared to 25 for ( H *) risk category (p < .0001). The ( L *) category, being the largest, was used as the reference level. Conclusion: This study provides the first real-world experience of using the IPSS-M and a modified version of the IPSS-M ( L, M, H) * risk category model to predict survival and progression into MN in CCUS (MUT) pt. The IPSS-M risk category was found to be a significant predictor of OS and MN-free survival. We propose that larger cohorts are needed to validate our findings.