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Abstract Background Missense mutations in IDH1 are well-established therapeutic targets in acute myeloid leukemia (AML). In contrast, the synonymous IDH1 variant p.Gly105Gly (rs11554137), present in 5–10% of AML cases, has been considered a benign polymorphism due to its high population frequency (5.7% overall, 0.7–10.5%) and lack of protein-altering effect. However, prior studies have associated this variant with inferior outcomes (Wagner, J Clin Oncol, 2010), though findings have been inconsistent. Few studies have examined its prognostic significance in the context of treatment intensity or allogeneic stem cell transplantation (SCT). Methods With IRB approval, clinical and genomic data were extracted from the Mayo Clinic AML database for patients diagnosed between Jan 1, 2018, and Jan 31, 2025. Patients were stratified by IDH1 Gly105Gly (rs11554137) status via NGS on bone marrow. Outcomes included co-mutation profiles, treatment response, measurable residual disease (MRD) by multiparametric flow cytometry, and survival. Multivariable Cox and logistic regression models for relapse-free survival (RFS) and overall survival (OS) were adjusted for age at diagnosis, ELN 2022 risk category, and SCT conditioning regimen. Results Among 379 AML patients, 36 (9.5%) harbored the IDH1 Gly105Gly (rs11554137) variant (cases) (median VAF 49%, IQR 48–49.2). In the full cohort, median age was 68 years (IQR 59–74), M:F ratio was 1.5; 75% had de novo AML, and median follow-up was 52 months. Among cases, common cytogenetic abnormalities included complex karyotype (28%), del(7q) (14%), and trisomy 8 (6%), while frequently mutated genes included DNMT3A (19%), IDH2 (17%), and BCOR (14%). Of 345 treated patients, 43% received intensive, and 56% received low-intensity regimens, including hypomethylating agent (HMA) alone (n=13), HMA+venetoclax (n=159), and targeted therapy (n=7). Among cases, complete remission (CR) rates were higher with intensive vs. low-intensity therapy (70% vs. 17%, p=0.03). MRD positivity post induction was more common in cases (66% vs. 26%, p=0.01), suggesting suboptimal leukemic clearance. Among 103 patients who underwent allogeneic SCT in CR1 (47% received myeloablative conditioning), morphologic relapse occurred in 64.3% of cases compared to 23.6% of controls (p = 0.004). Cases experienced significantly inferior median RFS (8 months [95% CI 5–NA] vs. not reached for controls, p <0.001), indicating more durable post-SCT remission in the control group. In a multivariable Cox proportional hazards model adjusting for age, ELN risk, conditioning intensity, MRD status, and complex karyotype, the IDH1 Gly105Gly variant remained independently associated with shorter RFS (HR 3.97, 95% CI: 1.74–9.05, p = 0.001). Complex karyotype was also an independent predictor of relapse (HR 3.35, 95% CI: 1.28–8.73, p = 0.014). Consistent with this, logistic regression revealed a significantly increased odds of post-SCT relapse among cases (OR 7.95, 95% CI: 2.19–35.27, p=0.001), independent of clinical covariates. Relapsed cases were significantly more likely to harbor FLT3-ITD mutations than relapsed controls (p=0.041). Pathway-level analysis revealed increased frequency of mutations in signal transduction genes (FLT3-ITD, CSF3R, NF1) (p=0.002). All CNS relapses among cases occurred in patients harboring at least one of these mutations. Despite the higher relapse rate, there were no significant differences in OS (14.4 vs. 11.3 months, p=0.36) between cases and controls. The IDH1 Gly105Gly variant was not independently associated with OS (HR 1.26, p=0.30). In contrast, age (p<0.0001) and adverse ELN risk (p<0.0001) were significant predictors of inferior OS. These findings suggest that the prognostic impact of IDH1 Gly105Gly is most pronounced in the post-SCT setting. Conclusion The synonymous IDH1 Gly105Gly variant (rs11554137) is an independent predictor of post-SCT relapse in AML, potentially mediated by co-occurring mutations in signal transduction pathways. Despite no observed impact on OS, likely due to effective salvage therapies, the variant's link to lower CR rates with low-intensity therapy and higher MRD burden suggests treatment intensity influences its clinical relevance. These findings support reevaluating the prognostic role of synonymous variants in AML and call for larger studies and mechanistic investigations into splicing, transcript stability, and translation.

Abstract Background Despite CD19-directed chimeric antigen receptor T (CAR-T) therapy achieving remarkable remission rates in relapsed/refractory B-cell acute lymphoblastic leukemia (R/R B-ALL), long-term survival remains constrained by T-cell exhaustion and immunosuppressive microenvironments. Dasatinib, a tyrosine kinase inhibitor (TKI), has been shown to alleviate T cell exhaustion and enhance CAR-T cell persistence, demonstrating therapeutic synergy in newly diagnosed ALL. However, it may also suppress CAR-T cytotoxicity and is ineffective against BCR-ABLT315I mutations. Third-generation TKIs, including ponatinib and olverembatinib, effectively overcome T315I-mediated resistance, yet their impact on CAR-T cell function and immune modulation remains insufficiently defined. Methods and Results: We first established in vitro co-culture models of CD19 CAR-T cells with the B-ALL cell line NALM6 to evaluate the effects of dasatinib, ponatinib and olverembatinib on resting and tumor antigen-activated CAR-T cells. At clinically relevant concentrations, none of the TKIs affected resting CAR-T viability. In contrast, upon activation, all three TKIs significantly inhibited CAR-T activation, cytotoxicity, and cytokine secretion (IL-2, TNF-α, IFN-γ), while reducing activation-induced CAR-T cell apoptosis. TKIs treatment also downregulated exhaustion markers (eg. PD-1, TIM3, LAG3) and promoted differentiation toward naïve (TN) and central memory (TCM) subsets, while reducing terminal effector T cells. After the drug elution, CAR-T cells regained their cytolytic function in response to tumor targets. Notably, ponatinib-treated CAR-T cells exhibited higher levels of IFN-γ, TNF-α and IL-2 post-washout compared to other groups. Importantly, the TN/TCM phenotype and low exhaustion marker expression were preserved after washout in all TKI-treated groups. Transcriptomic profiling of CAR-T cells in vitro revealed that all three TKIs downregulated key exhaustion-associated genes, including PDCD1, HAVCR2, CTLA4 and NR4A1, along with upregulation of stemness- and memory-associated genes (eg. CCR7, TCF7, LEF1, IL7R). Gene set enrichment analysis showed inhibition of T-cell activation, apoptosis, glycolysis, and TGF-β signaling by all TKIs. Remarkably, ponatinib uniquely activated the IFN-γ pathway, suggesting superior immunostimulatory effects. Mechanistically, all three TKIs suppressed the phosphorylation of AKT, p38 MAPK, and NF-κB p65, indicating shared inhibition of downstream TCR signaling. Based on these findings, we evaluated ponatinib in a clinical setting. A prospective study (No. ChiCTR2000036350) enrolled three R/R B-ALL patients with the BCR-ABLT315I mutation. All the patients received CD19 CAR-T cell therapy followed by ponatinib maintenance, initiated two months post-infusion. All three patients remained progression-free during ponatinib maintenance. Notably, the DNA copies of CAR-T cells remained detectable at months 5 and 9 after ponatinib treatment in two patients, respectively. To further explore the in vivo effects of ponatinib on T cells and other immune cells,peripheral blood mononuclear cells were collected from these patients before and one month after ponatinib initiation for single-cell RNA sequencing. Ponatinib exposure led to an increased proportion of CD8+T cells and a decreased abundance of myeloid cells subsets. Within T cells, effector cells expanded while Tregs and exhausted T cells decreased. Differential gene analysis showed suppression of activation, adhesion, apoptosis, and PD-1/PD-L1 signaling, aligning with in vitro results. Functional scoring revealed enhanced cytotoxicity, IFN-γ responses, and reduced glycolysis, exhaustion, TGF-β, and Treg signatures. Furthermore, analysis of NK cell subsets showed enhanced cytolytic scores and enrichment of proliferation and immune activation pathways, suggesting that ponatinib preserves innate immune responses in vivo. Conclusions: This study provides preclinical and preliminary clinical evidence that third-generation TKIs, particularly ponatinib, can effectively alleviate CAR-T cell exhaustion, promote memory phenotypes, and remodel the immune microenvironment. Compared to dasatinib and olverembatinib, ponatinib uniquely enhances effector cytokine production and activates the IFN-γ pathway. These findings support ponatinib as a promising combinatorial agent with CAR-T therapy, offering a rational strategy for improving outcomes in patients with T315I-mutated R/R B-ALL.

Abstract Introduction: Autologous CD19 CART therapies have been utilized across a wide range of B cell driven autoimmune diseases (AD), including systemic lupus erythematous (SLE), idiopathic inflammatory myopathies (IIM), systemic sclerosis (SSc), & myasthenia gravis (MG). To date, a number of autologous CD19-CART therapies have delivered durable drug free responses in AD patients. However, the safety profile of autologous CD19-CART therapies across different AD has yet to be fully explored. This is especially true for CRS & ICANS. Here, we provide novel insight into the mechanism underlying the safety profile of resecabtagene autoleucel (rese-cel) an autologous CD19 41BBz CART therapy across four separate Phase I/II clinical trials in SLE, IIM, SSc, & MG. Objectives: The primary objective of each trial is safety & tolerability of rese-cel at Day 29. Key secondary objectives include changes in clinical scores & use of immunomodulatory agents. Key translational assessments include CART cell pharmacokinetics (PK), impact on peripheral B cell populations (pharmacodynamics), & serum cytokine levels. Methods: 19 Patients (data cutoff: 6/2/2025 SLE; 5/6/2025 IIM, SSc, and MG), with active disease refractory to standard of care have been treated across 4 Phase I/II clinical trials in SLE (8 patients; NCT06121297), IIM (8 patients; NCT06154252), SSc (2 patients; NCT06328777), & MG (1 patient; NCT06359041). All patients were treated with a single weight-based infusion of rese-cel at a dose of 1 x 106 cells/kg following lymphodepletion (flu 25 mg/m2/d on Days -5, -4, & -3, & cy 1,000 mg/m2/d on Day -3). All non-glucocorticoid immunomodulatory agents were stopped prior to lymphodepletion. Glucocorticoids were tapered post-infusion. Patients were not given prophylactic tocilizumab pre-infusion. Translational assessments were determined as follows: CART PK was evaluated by flow cytometry & dPCR; B-cell enumeration & phenotyping were evaluated by flow cytometry; & serum cytokines were evaluated via immunoassay. Results: Across all indications (19 patients), 7 episodes of CRS were observed (7/19). 6 of 7 patients experienced a Grade 1 CRS, & 1 patient had a Grade 2 CRS. 2 patients developed ICANS; 1 patient had a Grade 4 (previously presented at ACR Convergence 2024), & the other patient had a Grade 3. Both ICANS events were rapidly resolved without sequelae using standard therapy. These CRS and ICANS events differed across indications. In IIM, 4 of 8 patients experienced Grade 1 CRS and no patients experienced ICANs. In SLE, 2 patients experienced Grade 1 CRS and one developed Grade 4 ICANs. In SSC, 1 of 2 patients experienced Grade 2 CRS & the other experienced Grade 3 ICANS. In MG, no CRS or ICANS was observed. Rese-cel infusion products were ~ 66% transduced, with a mean CD4:CD8 ratio of 6. Post-infusion, peak CART expansion was observed on day 12 & the mean peak expansion was 89 cells/µL (SD +/- 214). B cells were rapidly depleted from peripheral blood post-infusion, with nadir occurring at 13 days & repopulation beginning at 62 days post-infusion on average. Re-emergent B cells were largely of the CD24+CD38+ transitional naïve phenotype. Commensurate with B cell depletion, serum BAFF induction was observed, with a mean fold induction of 16 & mean peak level of 56,404 pg/µL (SD: +/- 45,547). Serum cytokines associated with CRS & ICANS; IFNg, IL6, & IL8 reached peak elevation on days 10, 9, & 10 respectively post-infusion. The mean peak elevation for IFNg, IL6, & IL8 were 232 pg/ µL (SD: +/- 320), 44 pg/ µL (SD: +/- 60), & 80 pg/ µL (SD +/- 73), respectively. Conclusion: Rese-cel appears to be well tolerated in autoimmune patients with rates of CRS & ICANS of 37% & 11% across all autoimmune indications, respectively (SLE patients had a 25% CRS rate & 12.5% ICANS rate, IIM patients had a 50% CRS rate & 0% ICANS rate, SSC patients had a 50% CRS & 50% ICANS rate, & MG patients had a 0% CRS & ICANS rate). Notably, the frequency & severity of CRS appears to be low, with no CRS in 63% of patients (12/19) & most CRS events (6/7) being Grade 1. The lower frequency & severity of both CRS & ICANS events are likely correlated with the relatively modest induction of serum IFNg, IL6, & IL8 found in autoimmune patients post rese-cel weight-adjusted dose infusion. High BAFF levels reflect deep systemic B cell depletion. Together, these data support further support late-stage clinical development of rese-cel in autoimmune disease.

Abstract Recent studies have highlighted the important but pleiotropic role of IL-4 in response to chimeric antigen receptor T (CART) cell therapy. IL-4 was identified as a regulator of dysfunction in CD19-targeting CART cells with a CD28 costimulatory domain (CART19-28ζ) through a genome-wide CRISPR knockout screen as well as RNA and ATAC sequencing of baseline and exhausted healthy donor CART19-28ζ cells and pre-infusion CART19-28ζ cell products from responders and non-responders in the ZUMA-1 trial (Stewart et al, Nat Commun., 2024). However, interrogation of a single-cell atlas fromactivated pre-infusion CART cell products from patients with acute lymphoblastic leukemia revealed a beneficial role for IL-4 and other type 2 cytokines in the promotion of long-term response (Bai et al, Nature, 2024). In this report, we aim to further understand how IL-4 can be detrimental to CART cell therapy. To study the regulation of CART19-28ζ cells by IL-4, we performed RNA sequencing on CART19-28ζ cells that were stimulated with CD19+target cells in the presence of either 20 ng/mL hrIL-4 or diluent for 24 hours, one week, or two weeks in an in vitro model for exhaustion as previously described (Stewart et al, Nat Commun., 2024). We observed that hrIL-4 treatment drove the development of a distinct transcriptomic profile after chronic stimulation for one week (up genes = 3253 and down genes = 3645) or two weeks (up genes = 6435 and down genes = 6224) but not following 24 hours of activation (up genes = 0 and down genes = 0). Gene set enrichment analysis on the differentially expressed genes following two weeks of chronic stimulation showed that hrIL-4 treatment pushes CART cells to a more exhausted and less naïve phenotype (FDR = 0.015) and leads to a broad downregulation in cytokine signaling (FDR = 0.012). To investigate transcription factors associated with IL-4-induced CART cell dysfunction, we used BETA analysis with publicly available transcription factor binding sites for STAT6, GATA3, and EOMES and our differential expression data. EOMES (p = 0.039), but not STAT6 (p = 0.999) nor GATA3 (p = 0.999) significantly regulated the differentially expressed genes. In support of these findings, the addition of hrIL-4 also significantly upregulated EOMES expression after one (p < 0.0001) and two (p < 0.0001) weeks of chronic stimulation and downregulated the expression of TBX21 (p < 0.0001), a binding competitor of EOMES, following two weeks of chronic stimulation. To evaluate if EOMES is critical for IL-4-induced CART cell dysfunction, we treated either control or EOMES CRISPR knockdown CART cells with 20ng/mL hrIL-4 during chronic stimulation. EOMES knockdown reduced IL-4-induced CART19-28ζ cell dysfunction as seen by an increase in antigen-specific expansion (p = 0.046) and an increase in cytokine production including IL-2 (p < 0.0001), TNF-α (p = 0.006), and IFN-γ (p = 0.020). Having identified that EOMES is at least partially responsible for IL-4-induced CART cell dysfunction, we evaluated EOMES binding sites in human CART19-28ζ cells with CUT&RUN sequencing. Our analysis revealed EOMES binding at hypersensitivity site 2 (HS2) in the IL-4 locus which, in a previous study, was more accessible in both exhausted healthy donor CART19-28ζ cells and pre-infusion CART cell products from non-responders in the ZUMA-1 clinical trial (Stewart et al, Nat Commun., 2024). To determine how HS2 can impact CART cell functions, we generated CART19-28ζ cells from either wildtype (HS2+/+) or HS2-deleted (HS2-/-) murine T-cells and tested them in a syngeneic model of lymphoma generated by engrafting C57BL6 mice with luciferase+ TBL12 cells (1x106, tail vein injection on Day -3) following preconditioning (250mg/kg cyclophosphamide, intraperitoneal injection on Day -4). Following randomization of mice via tumor burden (n = 5 per group), mice were treated with either HS2+/+ or HS2-/- CART19-28ζ cells (1x106 cells, tail vein injection). In this model, HS2-/- CART cells showed enhanced expansion (p = 0.022), increased anti-tumor activity (p = 0.018), and improved overall survival (p = 0.003). Together, our study shows that IL-4 induces a distinct cellular fate in CART19-28ζ cells in the presence of chronic antigen-specific stimulation at least partially through the upregulation of EOMES. Further, the epigenetic regulation of CART cells by the IL-4 enhancer HS2 can significantly influence CART cell activity, and deletion of HS2 can improve CART cell therapy.

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 BACKGROUND: DNA methyltransferase inhibition offers palliative benefit but does not alter disease biology. in chronic myelomonocytic leukemia (CMML, Merlevede J et al. Nat Comm 2016). This was conclusively demonstrated in the phase 3 DACOTA study, where decitabine did not improve event-free survival in comparison to hydroxyurea (median 12.1 versus 10.3 months, P=0.27) in advanced myeloproliferative CMML (MP-CMML). Hence, there is in urgent need to develop disease modifying therapies for this dismal condition. We and others have demonstrated a role for RAS mutations in MP-CMML through the activation of the KMT2A-PLK1 axis. Targeting of this axis using onvansertib, an inhibitor of PLK1, has demonstrated its pre-clinical efficacy in MP-CMML patient-derived organoid and xenograft models. (Carr RM et al. Nat Comm 2021). METHODS: We conducted a single center phase 1 study to assess the safety and efficacy of onvansertib in relapsed/refractory MP-CMML (NCT05549661). We used a Bayesian Optimal Interval (BOIN) study design to determine the maximum tolerated dose (MTD). The three dose cohorts included 6, 9 and 12 mg/m2 given orally from days 1 to 21 of each 28-day cycle. Key inclusion criteria included a WHO-defined diagnosis of MP-CMML with a white blood cell (WBC) count > 13,000/mm3 and adequate organ function (estimated GFR > 50 mL/min/m2). Response was evaluated per the MDS/MPN International Working Group Criteria (Savona et al. Blood 2015). Targeting efficacy was assessed by measuring phosphorylation of TCTP (a PLK1 substrate), specifically pTCTP/TCTP ratios by immunoblotting in peripheral blood mononuclear cell (PBMC) samples pre and post cycle 1 (Days 1, 7, 21) and end of cycle 3. Pharmacokinetic studies and translational correlative studies were conducted per protocol. RESULTS: Between April 2023 and July 2025, a total of 9 patients were enrolled. The median age at enrollment was 68 years (range: 60-83); all caucasian with 4 (44%) males. All patients had a diagnosis of CMML-1 with a median bone marrow (BM) blast percentage of 4 (0-9), median hemoglobin 10 (7.1-13.2) g/dL, median WBC count of 11.1 (3.1-72.7) x 109/L (4 patients had a WBC count < 13 x 109/L at enrollment due to hydroxyurea use but were confirmed to be MP-CMML at diagnosis), absolute monocyte count 2.1 (1.9-19.6) x 109/L, and platelet count of 36 (20-494) x 109/L. TET2 (89%), SRSF2 (78%), ASXL1 (56%), NRAS, KRAS and CBL (22% each) were the most frequent molecular abnormalities. Previous therapies included hydroxyurea (n=8) and decitabine (n=1). Four patients were enrolled at the 6mg/m2dose level, 2 at the 9 mg/m2 dose level and we currently have one patient at the 12 mg/m2 dose level. While there were 2 planned patients at the 6 mg/m2 dose level, due to a case of pericardial effusion that was possibly drug related, an additional two patients were enrolled at the same dose level, without any dose limiting toxicity (DLT). Targeting efficacy was confirmed by pTCTP immunoblotting in PBMC samples from 4 patients (6 and 9 mg/m2 dose levels) showing suppressed pTCTP/TCTP ratios. No confirmed DLT has been identified thus far, and no grade 3 or 4 toxicities attributable to the study drug have been reported. Possible or unrelated grade 3 adverse effects included infections (sepsis/cellulitis, n=2, 22%), acute kidney injury (n=1, 11%), pericardial effusion (n=1, 11%), and atrial fibrillation with rapid ventricular response (n=1, 11%). Median duration on therapy was 124 (range: 15-654) days with most common cause of study discontinuation being disease progression (33%). Response was assessed as per the MDS/MPN IWG criteria in patients who received at least 1 cycle (n=8) with 3 (38%) patients meeting criteria for either hematological or optimal marrow response. Specifically, there was one (14%) patient with >100% platelet response, one (14%) with normalization of WBC count (baseline WBC count >50 x 109/L), one (14%) optimal marrow response with BM blast% reduction from 8 to 2, facilitating candidacy for an allogeneic hematopoietic stem cell transplantation, and three patients (38%) remained as stable disease. CONCLUSION: In MP-CMML, PLK1 inhibition with onvansertib is relatively well tolerated and shows preliminary efficacy in approximately 40% of patients, with one patient having an optimal marrow response at the 9 mg/m2 dose. Future directions include a dose expansion phase and studying upfront combination with DNA methyltransferase inhibitors.

Abstract Introduction In 2018, our institution implemented a MALDI-TOF mass spectrometry (MS) method (Mass-Fix) to detect myeloma associated M-proteins in blood. Currently, the lab has performed more than 500K tests by Mass-Fix. While Mass-Fix has been found to be more sensitive and specific than IFE, an ESI-Q-TOF (High Resolution Mass-Fix) method has demonstrated the potential to detect M-proteins at significantly lower levels. Blood minimal residual disease (MRD) detection in lieu of bone marrow (BM) is desirable since blood is easier and less expensive to obtain and analyze. This feature can allow for more frequent MRD monitoring for myeloma patients. Methods 66 residual blood samples from myeloma patients with varying M-protein varying isotypes were diluted into healthy blood to estimate the limit of detection of HR-Mass-Fix method. 179 Rochester Mayo Clinic patients who had a clinically ordered BM MRD assessment by next generation flow (NGF) cytometry (2 x 10-6), a blood sample available within 7 days of the BM biopsy, and a pre-treatment measured M-protein mass by Mass-Fix were utilized to assess the concordance of the BM and blood MRD status. Results The average limit of detection (LOD) of the HR-Mass-Fix was found to be significantly lower than Mass-Fix (0.0003 g/dL and 0.01 g/dL, respectively). The treated patient cohort utilized for comparison consisted of 145 myeloma, 21 AL amyloid, 4 LPL lymphoma and 9 other PCDs. Comparing the Mass-Fix and HR-Mass-Fix results, 100% of blood positive MRD results by Mass-Fix were positive by HR-Mass-Fix. HR-Mass-Fix detected residual M-proteins (mass within +/- 1 Da of pre-treatment) in 29 samples (40%) that were negative by Mass-Fix. Thus, HR-Mass-Fix increased the overall blood MRD positivity rate from 57% by Mass-Fix to 78%. The BM NGF MRD positivity was lower at 54%. The overall agreement of blood and BM by Mass-Fix was 71% (kappa 0.4) which decreased to 0.63 (kappa 0.3) with HR-Mass-Fix. Most of the disagreements were HR-Mass-Fix Blood (+)/BM NGF (-). Blood M-protein MRD appeared to be affected by M-protein half-lives. To determine this, the M-protein isotype distribution of Blood/BM MRD status was compared the M-protein isotype distribution of the overall cohort. The half-lives are as follows: IgG 23 days, IgA 6 days, IgM 5 days and FLC 0.25 days. When both blood and BM were positive, the isotype distribution was not significantly different than the overall cohort. For blood(+)/BM(-) patients, IgG (with the longest half-life) were significantly overrepresented compared to the overall cohort (74% vs 51%) . For blood(-)/BM(+) cases, there were no IgG M-proteins, and the majority were FLC patients. These results suggest that patients with shorter half-life M-proteins (non-IgG) are more likely to be Blood MRD(-) than patients with longer half-life M-proteins. M-protein rate changes over time will likely be needed to resolve the M-protein half- life affect. Conclusions HR-Mass-Fix can significantly increase the detection of M-proteins in comparison to our current Mass-Fix method. Ig recycling was found to be a likely reason for the disagreement between BM and blood. Therefore, a negative blood MRD status by HR-Mass-Fix is most likely to be the most significant in predicting patient outcomes.

Abstract Introduction Understanding the tumor immune microenvironment (TIME) in multiple myeloma (MM) is critical for identifying prognostic biomarkers and therapeutic targets. We hypothesized that specific immune cell populations within the bone marrow TIME are associated with clinical outcomes and could enhance patient stratification. To this end, we conducted the largest CyTOF-based immune profiling study to date in MM, profiling >300 million bone marrow immune cells from patients enrolled in the CoMMpass study. Methods We analyzed >1,000 bone marrow samples from ~700 newly diagnosed MM patients enrolled in the MMRF CoMMpass study (NCT01454297), using a harmonized protocol and 41-antibody CyTOF panel at five academic centers. The majority were baseline samples (n=671), with additional samples spanning up to 3.5 years post-diagnosis (n>300). Each site contributed ~200 samples on average. The cohort included 40% females, with a median age of 63 years and BMI of 27.8. ISS stages were evenly distributed (I: 36%, II: 36%, III: 29%), and 84% had ECOG 0-1. Autologous stem cell transplant was performed in 58% of patients, and 22% received maintenance therapy. Results We profiled 311 million single cells across the cohort, identifying major immune compartments: Neutrophils (36.0%), CD4 T cells (19.7%), NK cells (9.9%), monocytes (7.6%), B cells (5.7%), and CD8 T cells (5.6%). Less abundant populations included dendritic cells, γδ T cells, plasmablasts, and pDCs. In our preliminary analysis with multivariate cox regression modeling, we identified clinical factors including stem cell transplant, ISS stage, and ECOG status, as independent predictors of overall survival (OS). After adjusting for age and sex, higher proportions of naïve B cells, pDCs, and γδ T cells were independently associated with improved OS, while CD4+ T cell abundance was positively associated with prolonged progression-free survival (PFS). In contrast, elevated neutrophil proportions were significantly linked to inferior OS and demonstrated a trend toward shorter PFS. Longitudinal immune profiling in more than 200 patients identified a gradual accumulation of naïve B cells and a pronounced post-treatment rise in CD45+ plasmablasts, highlighting persistent B cell activity as a candidate mechanism for sustained therapeutic efficacy. Conclusion This study presents the largest immune proteomic atlas in MM to date, linking TIME composition to clinical outcomes. A favorable immune landscape was marked by preserved humoral immunity (naïve B cells, plasmablast expansion), enrichment of pDCs and γδ T cells, and lower neutrophil abundance, collectively associated with improved survival outcomes. These findings lay the groundwork for immune-guided prognostication and highlight new opportunities for therapeutic targeting in MM. Ongoing analyses aim to further resolve baseline and longitudinal immune dynamics in relation to treatment response.

Abstract Background: Disease relapse is a major cause of mortality post allogeneic hematopoietic stem cell transplantion (alloHCT) in patients with myeloid neoplasms (MN). Cytogenetic risk classification is a key prognostic factor in predicting relapse post-transplant. Next generation sequencing (NGS) offers added prognostic insight by identifying relapse-associated mutations. We assessed the impact of mutation type and burden on post-alloHCT relapse risk and relapse-free survival (RFS) in MN patients with non-adverse-risk cytogenetics. Methods: We retrospectively reviewed patients who underwent alloHCT at Mayo Clinic Rochester between 01/01/2018 - 10/01/2023. Eligible patients had MN with non-adverse-risk cytogenetics [Acute myeloid leukemia (AML) with non-adverse-risk cytogenetics per ELN 2022, myelodysplastic syndrome (MDS) with non-high/very high-risk cytogenetics per IPSS-R criteria, and myeloproliferative neoplasms (MPN) (excluding myelofibrosis), chronic myelomonocytic leukemia (CMML), or MDS/MPN overlap with non-complex and non-monosomal karyotypes]. The primary endpoint was 3-year cumulative incidence of relapse (CIR) post-alloHCT, analyzed via competing risk models. RFS was estimated via Kaplan-Meier method. Prognostic mutations were identified using univariate (UVA) and multivariate (MVA) Cox models. A decision tree was used to define mutation-based relapse risk groups. Results: Of 585 patients, 215 (37%) met inclusion criteria; 133 (62%) were male. Median age at diagnosis was 61 years (IQR 53-65.5). Diagnoses included AML (125, 58.1%), MDS (55, 25.6%), CMML (17, 7.9%), MDS/MPN (11, 5.1%), and MPN (7, 3.3%). Median follow-up post-alloHCT was 3.2 years (IQR 1.9-5.2). Donor sources were mostly matched unrelated donors (120; 55.8%), followed by matched related donors (72; 33.5%), haploidentical donors (15; 7%), mismatched unrelated donors (6; 2.8%), and cord blood transplants (2; 0.9%). Most patients reduced intensity/non-myeloablative conditioning (144, 67%), while others received myeloablative (71, 33%). Mutation frequencies were as follows: ASXL1 58 (27.0%), RUNX1 45 (20.9%), FLT3 41 (19.1%), SRSF2 41 (19.1%), DNMT3A 39 (18.1%), STAG2 22 (10.2%), SF3B1 18 (8.4%),BCOR 18 (8.4%), WT1 16 (7.4%), U2AF1 13 (6.0%), ZRSR2 10 (4.7%), TP53 6 (2.8%), EZH2 3 (1.4%). In UVA, mutations in FLT3 (HR 2.15, p=0.018), SF3B1 (SF3B1 HR 3.08, p=0.002), PTPN11(HR 2.93, p=0.032), and WT1 (HR 2.29, p=0.069) were associated with increased risk of 3-year CIR. However, only FLT3 (HR 1.98, p=0.042) and SF3B1 (HR 4.0, p<0.001) retained significance in MVA. SF3B1 correlated with inferior 3-year RFS in UVA (HR 1.99, p=0.034) but not in MVA. Mutations in ASXL1, BCOR, EZH2, RUNX1, SRSF2, STAG2, ZRSR2, U2AF1, and TP53 had no significant impact on CIR or RFS. Harboring ≥2 or ≥3 mutations did not impact CIR or RFS. A decision tree model based on SF3B1, FLT3, DNMT3A, and WT1 mutations stratified patients into 3 risk groups. The high-risk group (n=32; 15%) had 50% 3-year relapse incidence (RI) and included patients with SF3B1 mutations or SF3B1-wildtype (WT) with concurrent FLT3 and DNMT3A mutations. The intermediate-risk group (n=36; 17%) included SF3B1-WT patients with either FLT3 mutations and DNMT3A WT (21% RI), or SF3B-1-WT and FLT3-WT patients with WT1 mutations (33% RI). The low-risk group (n=147; 67%) lacked all 4 mutations and had the lowest 3-year RI of 14%. The 3-year CIR significantly differed across these 3 risk groups (51.9%, 27.3%, and 15.8% in high, intermediate, and low-risk groups respectively; p<0.001). The 3-year RFS was significantly inferior in the high-risk group (35.55%, p=0.001), but similar among intermediate and low-risk groups (67.11% and 63.4%, respectively). Conclusion: In non-adverse risk cytogenetic MN, SF3B1 and FLT3 mutations predicted relapse post-alloHCT. Notably, mutations that were traditionally associated with adverse-risk AML per ELN 2022 (e.g, ASXL1, TP53) were not associated with relapse in this cohort. The discrepancy may reflect exclusion of patients with high-risk cytogenetics, as the prognostic impact of these mutations may depend on co-cytogenetic anomalies. In non-adverse-risk cytogenetics MN, we identified 3 groups of patients based on the presence or absence of SF3B1, FLT3, DNMT3A, and WT1 mutations. Those with mutated SF3B1 or concurrent FLT3/DNMT3A mutations experienced the highest risk of relapse post-alloHCT, translating into inferior RFS. Our findings need to be verified in a larger data registry.

Abstract Use of Direct oral anticoagulants (DOAC) has increased exponentially since their introduction in 2010. Over the years they have been used without any systematic oversight, there have been ongoing concerns related to inappropriate or incorrect dosing, incorrect duration, and non-adherence to the drugs leading to adverse patient outcomes. Several factors have been attributed to DOAC non-adherence such as presence or fear of adverse effects, affordability, lack of perceived benefit, or concerns for reversibility. These barriers impact both primary (first time prescription) and secondary adherence (longer term prescription). Poor adherence can lead to adverse outcomes such as stroke, death, and higher medical resource utilization hence increased cost to the patient and payors. This study was conducted as a quality improvement (QI) initiative to improve the adherence to the prescription DOACS. We included patients 18 years old, a diagnosis of Atrial fibrillation and flutter who had a primary care provider writing the prescription for chronic DOAC therapy. Patients with venous thrombosis, pregnancy or who were institutionalized were excluded. The charts were reviewed for primary and secondary adherence using Surescripts functionality which can provide baseline information about prescription fills from the participating pharmacies. In the preimplementation phase we identified 1030 patients meeting inclusion criteria associated with the pilot sites within Mayo Clinic practice in preceding 6 months. For this population, Surescripts data found that the rates of primary and secondary adherence to be 84% and 86% respectively. We then implemented a 6-month pilot where we created workflow for a pharmacy led DOAC management unit. Herein, once a DOAC prescription was made for patients meeting inclusion criteria, they would enter a registry where the pharmacy team would review the charts for primary adherence by utilizing Surescripts data. If adherent these patients would be marked for follow up in the secondary adherence phase. However, if non adherent, the team would initiate intervention including engagement with patient online services which was further escalated to phone calls by the pharmacy team. Once the barriers were identified several remediation approaches were incorporated in the process, these included education about DOACS, medicare education, social work assistance to even switching to warfarin if necessary. The same process was followed for secondary adherence management. In the intervention cohort, we had 411 patients during the pilot, for primary adherence 87.1% of patients were adherent at baseline and an additional 3.4% became adherent with interventions, while 9.5% remain nonadherent. The primary causes of nonadherence, amongst 9.5%, were cost (86%) while 1% were concerned about side effects and 3% were concerned about lack of perceived benefit. The inverntions lead to impoved adherence in most patients and very few patients were transitioned to warfarin. For secondary adherence 92% of the patients were adherent without any intervention, an additional 4% of patients benefited with the interventions of the DOAC management unit with approaches to address cost considerations, while another 4% remained nonadherent where cost remained the primary reason for nonadherence, other reasons being lack of perceived benefit and side effect concerns. In summary, with active intervention, in our patient population we were able to improve the primary adherence from 84% to 91% and secondary adherence from 86% to 96% . Cost remained the biggest barrier for adherence followed by concern for side effects and lack of perceived benefit. Our engaged team of pharmacy staff was able to manage those concerns with the education and cost mitigation strategies leading to improved adherence. Based on previously published literature even a 5% difference in adherence to DOACS can make a huge difference in population-based outcomes. Our work was able to demonstrate a positive impact of the intervention, highlighting the need for a well-managed DOAC unit especially for complex care populations.