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Importance The choice of induction agent during rapid sequence intubation (RSI) of critically ill adults may affect clinical outcomes. Although ketamine and etomidate are frequently used for RSI, their comparative effectiveness remains controversial. Objective To compare the safety of etomidate vs ketamine for emergency RSI in critically ill adults. Design, Setting, and Participants This cohort study used a target trial emulation with observational data collected between March 1, 2022, and April 30, 2024, from 18 emergency departments across Brazil, with data from the Brazilian Airway Registry Cooperation. Adults who underwent RSI and received either etomidate or ketamine as a sole hypnotic agent were included; those with preintubation cardiac arrest or immediate postintubation transfer were excluded. Exposure Administration of etomidate or ketamine as the induction agent for RSI. Main Outcomes and Measures The primary outcome was 28-day in-hospital mortality. Secondary outcomes included 7-day in-hospital mortality, first-attempt intubation success, and major adverse events (new hemodynamic instability, severe hypoxemia, and cardiac arrest) within 30 minutes after intubation. Inverse probability of treatment weighting was used to adjust for confounding. Risk ratios (RRs) and risk differences (RDs) with 95% CIs were used to compare outcomes between groups. Results Among 1810 patients (median age, 64 years [IQR, 50-74 years]; 1048 men [57.9%]), 514 received ketamine and 1296 received etomidate. The median shock index was higher in the ketamine group than the etomidate group (0.81 [IQR, 0.65-1.01] vs 0.76 [IQR, 0.59-0.99]), and preintubation vasopressor use was more common in the ketamine group than the etomidate group (191 of 514 [37.2%] vs 391 of 1296 [30.2%]). Weighted 28-day mortality was higher with etomidate than ketamine (60.5% [95% CI, 57.2%-63.8%] vs 54.4% [95% CI, 45.0%-63.9%]; RR, 1.14 [95% CI, 1.03-1.27]; RD, 7.6% [95% CI, 2.0%-13.3%]). Seven-day mortality was also higher with etomidate than ketamine (35.2% [95% CI, 32.0%-38.3%] vs 30.1% [95% CI, 23.5%-36.7%]; RR, 1.19 [95% CI, 1.04-1.35]). New hemodynamic instability within 30 minutes after intubation was more frequent with ketamine (24.2% [95% CI, 20.4%-28.0%] vs 18.9% [95% CI, 16.7%-21.0%]; RR, 0.78 [0.64-0.95]). There were no statistically significant differences in the other secondary outcomes. Conclusions and Relevance In this cohort study of critically ill adults undergoing RSI, etomidate use was associated with higher in-hospital mortality at 7 and 28 days compared with ketamine. These findings highlight the need for definitive randomized clinical trials to compare both agents.

Objective Posterior reversible encephalopathy syndrome (PRES) is a clinicoradiological diagnosis that can be associated with SLE. The clinical presentation, risk factors and outcomes of PRES in patients with SLE remain insufficiently described. Methods We conducted a multicentre cohort study at the Mayo Clinic and performed a systematic literature review (SLR) of patients with incident PRES and SLE. We described the clinical features, treatments and outcomes. Across both the cohort study and SLR, we assessed neurological sequelae, recurrence and survival. Within the cohort study, we examined PRES risk factors and discontinuation of antiseizure medications (ASMs). We used logistic regression to evaluate factors associated with neurological sequelae and Cox regression to assess predictors of recurrence. Kaplan-Meier methods estimated the rates of recurrence, survival and ASMs discontinuation. Results 550 cases (53 from the cohort study) were included, of whom 92% were female. Across both cohort and SLR, the most common symptom was seizure, occurring in 80%. PRES was the first manifestation of SLE in 13% of cases. In the adjusted analysis, older age was associated with neurological sequelae (OR 1.63 per 10-year increase; 95% CI 1.12 to 2.39). After 5 years of follow-up, 11.5% (95% CI 2.3 to 19.9%) of patients had a recurrent PRES episode. 30-day and 180-day survival following PRES was observed in 96.3% (95% CI 93.5 to 99.1%) and 93.7% (95% CI 89.8 to 97.7%) patients, respectively. In the Mayo Clinic cohort, 81% of PRES cases were attributed to hypertension in the context of lupus nephritis (LN), and by the fifth year of follow-up, 78.1% (95% CI 58.7 to 88.4%) of patients had discontinued ASMs. Conclusions In this cohort, most of the incident PRES episodes were preceded by hypertension and occurred in patients with LN. Although short-term and long-term prognoses are favourable, older patients more frequently experienced neurological sequelae.

Abstract Wiskott-Aldrich syndrome (WAS), an X-linked disorder characterized by immunodeficiency, thrombocytopenia, autoimmunity, and malignancy, can be effectively treated with allogeneic hematopoietic cell transplantation (HCT). Older age at HCT and mismatched donors are known to affect overall survival (OS). However, the influence of specific clinical manifestations or WAS variant class on OS and factors associated with event-free survival (EFS) remain incompletely defined. We analyzed outcomes of 308 patients with WAS who underwent HCT at 37 institutions of the Primary Immune Deficiency Treatment Consortium from 1990 to 2018. With a median follow-up of 5.3 years, the 5-year OS and EFS were 87.2% and 79.7%, respectively. Age ≥5 years, donor type, and a pre-HCT history of severe infection had a negative impact on OS and EFS, whereas pre-HCT autoimmunity had no impact. Reduced-intensity regimens were associated with lower T-cell and myeloid donor chimerism, particularly when non–busulfan-based regimens were used. Low myeloid donor chimerism was associated with lower platelet counts. Mixed chimerism was not consistently associated with post-HCT autoimmunity. Patients with class I (exon 1-2 missense and intron 5 hot spot variants) and class II variants (all others) had similar pre-HCT clinical symptom severity and no difference in OS, EFS, or platelet recovery post-HCT. In conclusion, our study showed excellent long-term OS and EFS after HCT for WAS, highlighting the importance of early HCT, before the development of severe infections. We confirmed that HCT using busulfan-based conditioning was associated with improved donor chimerism and platelet recovery. This trial was registered at www.clinicaltrials.gov as NCT02064933.

Abstract BACKGROUND Children exposed to poverty during frontline therapy for B-lymphoblastic leukemia (B-ALL) experience inferior overall survival (OS), higher relapse rates, and earlier relapse than their peers. Real-world data describing treatment patterns and survival after relapse of B-ALL are lacking and, as a result, the impact of poverty following relapse remains poorly understood. This study evaluated the association of poverty exposure, proxied by insurance status, with post-relapse survival and explored potential explanatory mechanisms, including disease biology and therapy utilization. METHODS Data from Retrospective Study of Contemporary Approaches to First Relapse in B-ALL (ReCALL-1), a multicenter retrospective cohort study of patients < 30 years old with first relapse of B-ALL from 2018-2022, were used. The primary exposure was poverty, proxied by insurance status at relapse, categorized as any private insurance (poverty unexposed), public-only insurance (poverty exposed), no insurance, or unknown; the latter two were not included in comparative analyses due to low numbers. The primary outcome was OS. Secondary outcomes included measurable residual disease (MRD)-negative second complete remission (CR2), relapse after CR2, non-relapse mortality (NRM), with relapse treated as a competing risk, and receipt of cellular and immunotherapies. RESULTS Among 462 patients from 31 U.S. institutions, 41% had private insurance, 47% public-only insurance, 2% no insurance, and 10% unknown. Compared to privately insured patients, those with public insurance were more likely to be Hispanic (56% vs 19%, p<0.001) and have non-English preferred language (30% vs 6%, p<0.001). Non-Hispanic Black patients comprised 9% of those with public insurance and 5% of those with private insurance. Disease characteristics that differed by insurance status at relapse included: Ph-like cytogenetics, more common in publicly insured (23% vs 11%, p=0.002), and infant ALL, more common in privately insured patients (10% vs 5%, p=0.06). Publicly insured patients were more likely to have experienced major organ toxicity during frontline therapy (32% vs 18%, p=0.002) and very early relapse (<18m from diagnosis: 43% vs 32%, p=0.07). Other demographic and disease characteristics, including relapse site and cytomolecular features, were similar by insurance status. With a median follow-up of 51 months from relapse, 4-year OS differed by insurance (log-rank p=0.02): 72% for private vs 56% for public insurance. After adjusting for race/ethnicity and language, public insurance increased the hazard of death by 65% (aHR 1.65 95% CI 1.13-2.42, p=0.004). The association remained, but was attenuated, when unbalanced disease characteristics (time to relapse, infant ALL, Ph-like cytogenetics, prior organ dysfunction) were included (aHR 1.47, 95% CI 1.00-2.16, p=0.05). Rates of MRD-negative CR2 were similar (85% public vs 87% private). However, point estimates suggested publicly insured patients had more relapse after CR2 (44% vs 37%, p=0.42) and higher 4-year NRM (16% vs 10%, Gray's test p=0.12), though neither were significant. Treatment choices for first relapse did not differ by insurance status. Utilization of intensive reinduction (91% vs 95% for public vs private, p=0.18) and clinical trial enrollment (22% for both) were similar. Receipt of cellular therapies did not significantly differ by public vs private insurance: Chimeric Antigen Receptor-T cells (CAR-T: 38% vs 34%, p=0.41), hematopoietic cell transplant (HCT: 42% vs 47%, p=0.32). Use of blinatumomab trended higher in those with private insurance (36% vs 45%, p=0.07), and inotuzumab was similar (11% for both, p=0.87). Median time from relapse to CAR-T or HCT was comparable (to CAR-T 102 vs 100 days, to HCT 117 vs 114 days). CONCLUSION This is the first study to demonstrate differential survival by insurance type, a proxy for poverty exposure, after first relapse of B-ALL. In this large multicenter, real-world cohort, patients with public insurance had worse OS after relapse than those with private insurance. The survival disparity existed independent of other SDOH factors. Adjustment for known high-risk disease features only partially accounted for the observed differences in OS. In contrast, rates of MRD-negative CR2 and receipt of CAR-T and HCT were comparable across insurance types. These findings highlight the urgent need to identify effective interventions that will ensure equitable outcomes after relapse.

Abstract Background Acute graft-versus-host disease (GVHD) is a major cause of non-relapse mortality (NRM) after allogeneic hematopoietic cell transplantation (HCT). High-dose steroids are standard first-line therapy for grade II–IV acute GVHD, whereas systemic treatment (tx) is generally not recommended for grade I. However, in practice many patients (pts) with grade I GVHD receive systemic steroids. A prior GITMO randomized trial (n=173) of grade I GVHD to either systemic steroid tx or “watch and wait” found that preemptive steroid tx reduced progression to grade II but not grade III/IV GVHD, increased serious infections, and resulted in similar chronic GVHD, NRM and survival (Bacigalupo, Haematologica 2017). In this study we analyzed real world evidence for early systemic steroid use and the potential impact of serum biomarkers in grade I GVHD. Methods We analyzed clinical data and serum samples from pts with grade I acute GVHD at 24 Mount Sinai Acute GVHD International Consortium (MAGIC) centers between 2014 and 2022 who were treated according to physician discretion. Pts were divided into a preemptive steroid tx group or a watch-and-wait group (systemic steroids administered only if progression to grade II–IV GVHD). Preemptive steroid use was treated as a time-dependent covariate and a frailty proportional hazards model with random effects was used to adjust for center effects to account for possible heterogeneity in clinical practices across transplant centers. Serum samples collected at the diagnosis of grade I GVHD were analyzed for ST2 and REG3α to calculate validated Ann Arbor (AA) scores (1, 2, 3). Results Grade I GVHD was diagnosed in 1143 pts; 591 (52%) received preemptive tx and 552 (48%) were treated by a watch-and-wait strategy. In the preemptive tx group, steroids were initiated at a median of 0 days and within 7 days for >90%. The median initial dose of steroids was 1 mg/kg of prednisone or its equivalent. In the watch and wait group, only 184 pts (33%) required systemic steroids for progression to grade II-IV GVHD at a median of 14 days from grade I diagnosis. Preemptive steroid tx reduced the risk of grade II–IV acute GVHD (HR, 0.71; 95% CI, 0.56–0.89; P=0.003), but not the risk of grade III/IV acute GVHD (HR, 1.03; 95% CI, 0.69–1.52; P=0.899) or chronic GVHD (HR, 1.03; 95% CI, 0.69–1.52; P=0.899), consistent with the GITMO trial. The 12-month incidence of NRM was higher for preemptive tx vs. watch and wait (13% vs 7%, P=0.002). In multivariate analysis preemptive steroid use independently predicted an increased risk of NRM (HR, 1.63; 95% CI, 1.06–2.50; P=0.026) as did several other known risk factors (older recipient age, umbilical cord blood donor, HCT-CI ≥3, and myeloablative conditioning). Greater NRM in the preemptive tx group was driven primarily by 3-fold more infectious deaths; deaths due to uncontrolled GVHD were similar for both tx groups. The risk for relapse was unexpectedly lower in the preemptive tx group (HR 0.64; 95% CI 0.47-0.88; P=0.006) and overall survival was therefore not different between groups, but risk factors for relapse such as minimal residual disease status were not available precluding further analysis of this finding. Serum was collected in 871 (76%) pts at onset of grade I GVHD for retrospective biomarker analysis. AA scores were AA1 (80%), AA2 (15%), and AA3 (5%). AA scores were highly predictive of progression to grade III/IV GVHD (AA1 vs AA2 vs AA3: 9% vs 14% vs 44%, all P<0.001) and the risk of 12-month NRM (8% vs 16% vs 39%, all P<0.001); this pattern was observed in both tx groups. The negative effect of preemptive steroid tx was most pronounced in pts with grade I/AA1 GVHD who experienced 5-fold more infectious related deaths compared to pts managed with a watch and wait approach. Conclusion Treatment of grade I GVHD with systemic steroids did not reduce the incidence of grade III/IV GVHD or deaths due to uncontrolled GVHD and instead increased the risk of NRM from infections compared to pts who were treated only after GVHD progressed. Importantly, 2/3 of the watch and wait pts never needed systemic steroid tx. These findings remained robust after adjusting for multiple confounders, including center effects, and were consistent with the GITMO trial. MAGIC biomarkers at the onset of grade I GVHD predicted outcomes and a watch and wait strategy may be best for the 80% of pts with AA1 GVHD. Better treatment options are needed for the 20% of pts with grade I and AA2/3 GVHD.

Abstract Introduction: cBTKi have made a substantial impact in pts w/ MCL, but pts who develop PD while on treatment w/ cBTKi have historically had poor responses to subsequent lines of therapy (LOT). Brexucel, lisocel, and the non-covalent BTKi pirto have been approved in this population. While two RW studies have substantiated the benefit of brexucel, similar data for pirto are lacking. Accordingly, we aimed to evaluate the outcomes of pts treated w/ pirto in the RW setting. Methods: We performed a multi-center retrospective cohort study of adult pts w/ a diagnosis of MCL who experienced PD after any approved cBTKi and subsequently received pirto. Pts who received pirto w/ bridging intent and those who switched to pirto due to intolerance of cBTKi were excluded. The 95% exact binomial confidence intervals (CI) were calculated for overall response rate (ORR) and complete response rate (CRR). Progression-free survival (PFS) and overall survival (OS) were defined as the time from pirto start to PD or death, whichever occurred first, and the time from pirto start to death, respectively, and calculated w/ the Kaplan-Meier method. The log-rank test was used to compare survival curves. A multivariate (MV) Cox proportional hazard model was built to calculate adjusted hazard ratio (aHR). Analyses were performed w/ SAS, version 9.4. Results: Data on 111 pts w/ MCL treated across 21 institutions were collected. Median (mdn) age was 72 (range 33 – 93) years. Twenty-seven pts (24%) were female, 3 (3%) were Asian, 5 (5%) Black, 9 (8%) Hispanic, 86 (77%) White. Among the 53 (48%) pts w/ available MIPI-c at diagnosis, 28 (53%) pts were high-risk. Ki67 at diagnosis was available in 84 (76%) pts: of those, 58 (69%) had a Ki67 ≥30%, and 41 (49%) ≥50%. TP53 mutational status was available in 70 (63%) pts: 33 (47%) had documented TP53 mutation and 10/52 (19%) pts had a disease w/ complex karyotype. Pts had a mdn of 3 (range 1 – 9) prior LOT (n=106). Ninety-one pts (86%) received chemo-immunotherapy (CIT), 16 (15%) autologous stem cell transplant, 19 (18%) venetoclax (ven), 29 (27%) chimeric antigen receptor T-cell, 4 (4%) allogeneic stem cell transplant. For cBTKi, 39 (35%) pts received ibrutinib, 56 (50%) pts acalabrutinib, 33 (30%) pts zanubrutinib, and 17 (15%) pts received more than one cBTKi. The mdn time from the last dose of cBTKi to pirto start was 55 (range 0 – 2587) days, w/ 51 (46%) pts who switched from a cBTKi directly to pirto. Thirty (27%) pts had no response (stable disease or PD as best response) to cBTKi, and 40 (36%) pts had no response to the last LOT. Two (2%) pts received pirto in combination w/ CIT, 8 (7%) w/ an anti-CD20 monoclonal antibody (mAb), 11 (10%) w/ ven ± a mAb, while 86 (77%) pts as single agent. In 104 (94%) pts, pirto was given at full dose, 10 (9%) pts had a subsequent dose reduction. A total of 35 serious (grade ≥3) adverse events (AE) were documented in 27 (24%) pts: 4 (4%) pts had a serious bleeding AE, 5 (5%) pts a serious cardiac AE, 10 (9%) a serious GI AE, and 16 (14%) a serious infectious AE. Ninety-eight (88%) pts were evaluable for response. The ORR was 40% (95% CI 30% – 50%) w/ a CRR of 29% (95% CI 20%– 39%). At the last follow-up, 29 (26%) pts remained on pirto w/ a mdn treatment duration of 15.1 months (range 0.4 – 26). Fifty-eight (52%) pts died, 35 (78%) because of PD. At a mdn follow-up of 15.8 (range 0.4 – 26) months among survivors, the mdn PFS was 4.7 months (95% CI 3.3 – 8.2) and the mdn OS was 13 months (95% CI 9.2 – not available). Using the log-rank test, age (quartile groups), number of LOT (1-2 vs. 3-4 vs. 5+), time from cBTKi to pirto (quartile groups), Ki67≥30%, and high MIPI-C did not result in a statistically significant difference in PFS (p>0.05). Presence of TP53 mutation was associated w/ a non-statistically significant adverse trend in PFS (p=0.0585). No response to cBTKi and Ki67≥50% were associated w/ a statistically significant inferior PFS in log-rank test (p=<0.05), which persisted in a MV Cox regression model (aHR 2.4, 95% CI 1.4-4.2, and aHR 2.6, 95% CI 1.6-4.4, respectively). Conclusions: This is the first RW report on pirto in MCL pts. In this high-risk population, ORR to pirto was comparable to the BRUIN trial, while PFS was significantly worse, especially in pts w/ no response to cBTKi and w/ a high Ki67. However, prolonged remissions were seen in a subset of pts, suggesting that a better characterization of this population w/ durable benefit from pirto is needed.

Abstract Introduction: Waldenström macroglobulinemia (WM) is characterized by the presence of a MYD88 mutation in >90% and a CXCR4 mutation in 30-40% of cases. The impact of these mutations on treatment responses and disease outcomes is well known. TP53 alterations (TP53ALT) have more recently been described in WM and are reported to occur in up to 25% of patients, with a higher frequency in patients with previously treated WM, especially those with prior alkylator or nucleoside analogue exposure (Tam et al Blood Adv 2024; Poulain et al CCR 2017; Tsakmaklis et al, ASH 2025). TP53 ALT WM is associated with a shorter time to disease progression, as well as worse overall survival (Poulain et al CCR 2017; Gustine et al, BJH 2019). Depth of response to BTK inhibitors is affected by TP53 status, with recent data reporting a very good partial response (VGPR) rate of 34% v. 25% and major response rates of 84% v. 73% in patients without or with a TP53 ALT (Tam et al Blood Adv 2024). No complete responses were reported and median PFS was shorter in those with a TP53ALT compared with wild-type (TP53WT). Loncastuximab tesirine is a humanized IgG1 anti-CD19 antibody that is conjugated to a pyrrolobenzodiazepine (PBD) dimer. Following internalization, the PBD dimer is released via proteolytic cleavage and binds to the DNA minor groove forming highly cytotoxic DNA inter-strand crosslinks. Cells deficient in DNA repair including TP53 alterations are vulnerable to the cytotoxic effects of loncastuximab tesirine (Tarantelli et al, Haematologica 2024). Methods: In an ongoing investigator-initiated, prospective phase II study in symptomatic patients with relapsed or refractory WM (NCT05190705), patients are receiving single agent loncastuximab tesirine. Loncastuximab tesirine is administered at 150 ug/kg in cycles 1 and 2 and 75 ug/kg in cycles 3 to 6. Each cycle is 28 days. The primary outcome measure of this study is attainment of VGPR or complete (VGPR/CR) response. The accrual goal of this trial is 20 patients, based on an alternative VGPR/CR rate ≥40% versus a null of ≤10%, with a double-sided alpha of 0.037 and a power of 87%. The study would meet its endpoint if ≥6 patients attained a VGPR/CR. Results: Of the 14 patients who have completed therapy on this trial to date, the median age is 71 years (range 53-81), 9 (64%) are male, and the median prior number of therapies is 4 (range 2-10). Median baseline hemoglobin is 9.5 g/dL (range 8.2-12.6), serum IgM is 2146 mg/dL (range 723-5955), and bone marrow involvement is 53% (range <5-90). Four patients had extramedullary disease. Twelve patients (86%) are MYD88 mutated; 8 patients (57%) are CXCR4 mutated, and 8 (57%) are TP53 altered. Nine patients completed all 6 cycles of therapy. Of the 5 other patients, treatment was stopped early due to GGT elevation (n=2), disease progression (n=1), patient decision (n=1), and skin toxicity (n=1). No IgM flare and no infusion reactions have occurred. For all patients, the median time to minor response, major response, and deepest response was 28, 55, and 172 days, respectively. The response rates include 1 (7%) CR; 9 (64%) VGPRs; 2 (14%) partial responses (PR); 1 (7%) minor response; and 1 (7%) progressive disease, for an overall response rate of 93% and a VGPR/CR rate of 71%. For the 8 patients with TP53ALT, the baseline characteristics were as follows: 50% male; median age of 70 years (range 53-77), median prior therapies is 4 (range 2-10); median IgM is 1877 mg/dL (range 723-5639); and median hemoglobin is 9.2 g/dL (range 8.2-11.6). All TP53ALT patients had an MYD88 mutation, and 6 (75%) carried a CXCR4 mutation. For those with TP53 ALT, the median time to minor response, major response, and deepest response was 28, 49, and 235 days, respectively. All TP53ALT patients attained a major response; 1 patient (13%) with a CR, 6 (75%) with a VGPR, and 1 (13%) with a PR, for an overall response rate of 100% and a VGPR/CR rate of 89%. One patient had splenomegaly and one had adenopathy, both of which resolved after treatment. Conclusion: Our findings from this ongoing, prospective trial demonstrate a high VGPR/CR rate of 71% in heavily pre-treated WM. Notable, is the VGPR/CR rate of 89% in high-risk patients with TP53ALT WM. No unexpected toxicities have been seen in this trial. Our findings identify a potentially novel time-limited therapy in relapsed or refractory WM, including patients with high-risk TP53ALT WM.

Abstract Nucleophosmin mutations are among the most common aberrations in acute myeloid leukemia (AML). We previously uncovered a novel pivotal role for mutant NPM1 (NPM1c) in the nucleus, where it binds chromatin at key self-renewal gene loci, such as HOXA/B and MEIS1, directly regulating oncogenic transcription. How NPM1c regulates the transcriptional activity of its chromatin targets remains unclear. To identify potential nuclear cooperating partners of NPM1c, we employed TurboID proximity labeling of NPM1c and wild-type NPM1 (NPM1wt). Mass spectrometric analysis revealed an enrichment of nucleoporins in the NPM1c interactome, with Nucleoporin 98 (NUP98) emerging as a top candidate. NUP98, a key component of the nuclear pore complex (NPC), has been established as a critical oncogenic player in AML as part of oncogenic fusion proteins. In these oncofusions the N-terminal FG (phenylalanine-glycine) repeat domain of NUP98 is preserved and fused to a diverse set of partner genes. NUP98-fusion partners are believed to mediate the chromatin binding specificity of the oncoprotein while the NUP98 FG repeats have been shown to form biomolecular condensates that enhance transcriptional output. Interestingly, wild-type NUP98 (NUP98wt) itself has been linked to gene regulatory functions during embryonic development and normal hematopoiesis. However, the role of NUP98wt in transcriptional regulation in the context of leukemia remains unresolved. To explore a potential cooperation between NUP98wt and NPM1c we first validated their interaction by co-immunoprecipitation assays. We next wanted to determine whether NUP98 and NPM1c are recruited to the same chromatin sites. To this end, we performed ChIP-seq in an NPM1c endogenous degrader cell line. These experiments revealed co-occupancy of NUP98 at NPM1c target genes and a significant reduction of NUP98 binding upon degradation of NPM1c. These findings suggest that NPM1c is required for the recruitment of NUP98 to key oncogenic target loci. To dissect the functional role of NUP98wt in NPM1c AML, we performed loss of function studies. CRISPR mediated knock-out of NUP98 in NPM1 mutant (OCI-AML3) cells resulted in a significant repression of NPM1c target genes, including HOXA/B and MEIS1. Given the essential nature of NUP98, we also engineered an endogenous NUP98 degrader system in OCI-AML3 and NPM1wt (MV4-11) cells to allow for the study of the effects of rapid protein depletion. Upon degradation of NUP98, OCI-AML3 cells exhibited robust myeloid differentiation (~70% increase in CD11b) and increased apoptosis (~50% increase in Annexin V+). MV411 cells on the other hand did not show the same differentiation phenotype. To characterize the immediate transcriptional consequences of NUP98 depletion, we performed nascent RNAseq after 1 and 3 hours of degradation. In the first hours of degradation we observed a rapid and significant repression of NPM1c target genes such as HOXA/B cluster genes, MEIS1, SMC4, TNRC18, SATB1/2 and UNCX, thus demonstrating the strong dependency of the NPM1c-driven transcriptional program on the presence of NUP98. In contrast, in MV4-11 cells, NUP98 loss had no effect on HOXA/MEIS1 expression, suggesting a leukemia subtype-specific role of NUP98wt. To delineate the domains of NUP98 required for its chromatin function, we performed a CRISPR gene tiling screen of full-length NUP98 (635 sgRNAs) and based on this we generated several domain-deletion mutants for rescue experiments in the NUP98-degrader cell lines. Retroviral overexpression of the N-terminal FG-repeat containing domains of NUP98 was sufficient to rescue cell viability and prevent the differentiation phenotype typically observed upon NUP98 degradation. Strikingly, gene expression analysis showed that the NUP98 FG-repeats were sufficient to restore the expression of HOXA10, MEIS1, and HOXB9 expression. Overall, our study identifies NUP98wt as a critical cofactor for NPM1c-driven oncogenic transcription. NPM1c recruits NUP98 to its chromatin targets, functionally substituting for an oncogenic fusion partner to concentrate transcriptional condensates at oncogenic loci. This establishes a previously unrecognized, subtype-specific dependency on NUP98wt in leukemogenesis.

Abstract Somatic mutations in hematopoietic stem and progenitor cells (clonal hematopoiesis) are known to alter the immune milieu and can result in autoimmune disorders including VEXAS (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) syndrome. There are currently no curative strategies for CH-linked autoimmune disorders besides allogenic hematopoietic stem cell transplantation. Recent work has shown a high prevalence of autoimmune syndromes among patients with IDH1/2 mutant myeloid neoplasms (MN). We thus hypothesized that IDH1/2-mutant CH might drive a subset of autoimmune disorders. To further characterize a possible association between IDH-mutant CH and autoimmune disease, we used data from two observational cohort studies, the UK Biobank (N=454,218) and All of Us (N=414,830). We observed 264 individuals with IDH1/2-mutant CH. IDH1/2 CH was associated with autoimmune disease (OR=1.8, p=4e-3), and, when compared to other CH genes with at least 100 carriers, showed the strongest effect size. When comparing autoimmune subtypes, we observed significant associations with polymyalgia rheumatica (OR=4.0, p=1e-3) and giant cell arteritis (OR=8.1, p=1e-6). IDH1/2 CH carriers had elevated CRP levels compared to other CH-carriers (p=1e-2) and non-carriers (p=8e-3). IDH1/2 CH was associated with lower neutrophil counts and higher monocyte counts. While most (68%) had blood counts outside the normal range, only a minority (11%) had severe cytopenias that met criteria for CCUS. Since IDH1/2 CH is associated with both autoimmune disease and blood count abnormalities, we sought to define the frequency, clinical presentation, and outcomes of IDH1/2-associated autoimmune disease among individuals with bone marrow biopsy confirmed CCUS. We used data from three CCUS cohorts: first, a multi-institutional cohort of CCUS patients with systemic autoinflammatory disease (SAID) who were UBA1-negative, second, a hospital-based cohort at Mayo Clinic, and third, a cohort of IDH1 carriers enrolled on a phase II clinical trial of ivosidenib (NCT05030441). Within 193 SAID patients who were free of myeloid neoplasm at the time of recruitment, IDH1/2 pathogenic variants were the most observed CH driver, occurring in 2% of individuals. These individuals (N=4) all had recurrent fevers and dermatologic manifestations, requiring chronic glucocorticoid treatment with 75% having inflammatory arthritis requiring multiple Disease-Modifying Antirheumatic Drugs (range 1-7, median=5). Within the Mayo Clinic cohort, 275 patients with CCUS were serially ascertained. Among those 8 (2.9%) were IDH1-mutant of whom 50% had autoimmune disease (three with seronegative rheumatoid arthritis and one with Sweet syndrome). At last follow up (median 16 months), 2 (25%) of 8 patients had progressed to MN. Within our open-label decentralized trial of ivosidenib (IDH1 inhibitor) in CCUS, we enrolled 20 patients, of whom 5 (25%) had a diverse array of pre-existing autoimmune disorders, including two cases of seronegative rheumatoid arthritis and one case each of Sweet syndrome, polymyalgia rheumatica, and Sjögren's syndrome. Underlying the clinical presentation of all five cases was episodic joint pain, with half also experiencing episodic dermatologic manifestations. The majority (58%, 11/19) of the IDH1mt-CCUS patients had an elevated hsCRP (>=5; range 6.91–46.2) including all four with co-occurring autoimmune disorders (range 7.97–46.2). Out of the 20 enrolled patients, 17 received at least 8 weeks of therapy, of which 9 had elevated hsCRP at baseline. Most of these (7/9) had normalization of hsCRP (falling below 5 mg/L) while on treatment (median time to hsCRP normalization of 58 days). Among the four patients with autoimmune disease, all had subjective improvement in their autoimmune symptoms following treatment with ivosidenib. In conclusion, we show that IDH1/2 CH is associated with autoimmune disorders and is particularly enriched among IDH1/2 CH carriers with co-occurring cytopenias. IDH1/2-associated autoimmune disease can range from classic rheumatic conditions such as rheumatoid arthritis to more severe VEXAS like presentations. IDH inhibition demonstrates efficacy for improving inflammatory manifestations within an ongoing clinical trial. Together these results nominate IDH mutant CH as a driver of autoimmune diseases with therapeutic implications.