Ras proteins are functionally dependent on one or more lipid modifications.1,2 The dynamic palmitoylation of N-Ras by DHHC palmitoyl acyltransferases and depalmitoylation by ABHD17 serine hydrolases is essential for the growth of NRAS-mutant acute myeloid leukemia (AML) cells.3-6 Here we show that ABD778, an in vivo-active ABHD17 inhibitor, selectively reduces the growth of NRAS-mutant AML and melanoma cell lines and is synergistic with the MEK inhibitor PD0325901 (PD901; mirdametinib). Mechanistically, ABD778 and PD901 induce deep and durable suppression of mitogen activated protein kinase (MAPK) pathway activation. Co-treatment extended the survival of mice transplanted with NrasG12D AMLs, which acquired by-pass mutations at relapse that conferred drug resistance and restored MAPK activation. ABD778 augmented the anti-leukemia activity of PI3 kinase, pan-Ras tri-complex, and FLT3 inhibitors, and restored gilteritinib sensitivity in a patient-derived xenograft model of FLT3 inhibitor resistance. These studies validate the palmitoylation cycle as a therapeutic target in NRAS-mutant cancers.

BCR::ABL1 acquisition is an emerging but poorly characterized resistance mechanism in FLT3-mutated AML. Using multiomic single-cell DNA sequencing, we characterized clonal evolution in a patient with FLT3-ITD AML who acquired BCR::ABL1 with FLT3 inhibitor resistance. Phylogenetic reconstruction confirmed BCR::ABL1 as a branch event co-occurring with FLT3-ITD and restricted to specific blast populations, supporting BCR::ABL1 acquisition as a resistance mechanism.

Frameshift mutations in exon 12 of nucleophosmin 1 (NPM1 mut) are among the most common mutations in acute myeloid leukemia (AML) and have historically been considered favorable-risk in the absence of FLT3-ITD. In the European LeukemiaNet (ELN) 2024 risk-classification for patients treated with hypomethylating agents plus venetoclax (HMA + VEN), NPM1 mut is not considered favorable when co-occurring with signaling gene (SG) mutations (i.e., FLT3-ITD, NRAS, KRAS). However, due to limited numbers in the original analysis, the prognostic impact of SG mutations in NPM1-mutant AML remains unclear. We evaluated the prognostic significance of NPM1 mut with and without SG mutations in two independent cohorts of patients ≥ 60 years with ELN 2024 favorable- or intermediate-risk AML treated with HMA + VEN. Cohort 1 included 322 patients treated in the academic setting. NPM1 mut (n = 61) was associated with a nonsignificantly longer overall survival (OS) compared to NPM1 wild-type (NPM1 wt) (median, 53.05 vs. 17.03 months, p = 0.10). In multivariable analysis (MVA), SG mutations were not independently prognostic within the NPM1 mut subgroup. Cohort 2 included 816 patients from a real-world community-treated cohort. NPM1 mut (n = 124) had a longer OS compared with NPM1 wt (median, 15.3 vs. 14.4 months, p = 0.03). In MVA, NRAS, KRAS, and FLT3-ITD were independent unfavorable prognostic factors; NPM1 mut with, compared to without, SG co-mutation had a shorter OS (median, 9.4 vs. 31.6 months, p = 0.001). These findings suggest SG mutations negate the favorable impact of NPM1 mut in older patients treated with HMA + VEN. Prospective clinical trials are needed to investigate the use of combination therapies to improve outcomes in this high-risk subgroup.

FLT3 inhibitor efficacy in AML with FLT3-ITD is short-lived, frequently due to new mutations, most commonly in NRAS. Sphingosine kinase 1 (SPHK1), which phosphorylates sphingosine to generate sphingosine-1-phosphate (S1P), is upregulated and localized to the plasma membrane in RAS-mutated cells. We studied S1P and FLT3 co-targeting to overcome FLT3 inhibitor resistance in NRAS-mutated FLT3-ITD AML cells. NRAS-mutated FLT3-ITD AML cell lines and patient blasts were treated with FLT3 inhibitors and/or S1P receptor (S1PR) modulators. FLT3 inhibitor sensitivity was assessed by immunoblotting, cytotoxicity, apoptosis and colony formation. Co-treatment was also assessed in vivo in an orthotopic mouse model. Downstream RAS and SPHK1 effectors were measured by immunoblotting and qRT-PCR. The S1PR modulators fingolimod (FTY720) and mocravimod (KRP-203) resensitized FLT3-ITD-expressing MOLM-14 and MV4-11 human AML cells with G12D, G12S, Q61K or Q61H, but not G12C, and patient blasts with G13D, G13V or G12D NRAS mutations to FLT3 inhibitors. Moreover, FTY720 co-treatment resensitized G12D NRAS-mutated M14(R)701 cells to gilteritinib in vivo. Co-treatment inactivated ERK, transcriptionally downregulated SPHK1, and inactivated downstream AKT, p70 S6K and BAD, with inactivation abrogated by constitutive SPHK1 expression. The clinically applicable S1PR modulators fingolimod and mocravimod resensitize NRAS-mutated FLT3-ITD AML cells to FLT3 inhibitors, supporting potential clinical efficacy.

Abstract Acute myeloid leukemia (AML) is a malignant hematopoietic disorder characterized by the clonal proliferation and impaired differentiation of myeloid progenitor cells. It is the most common type of acute leukemia in adults and is associated with high rates of chemotherapy resistance and relapse. Mutations in the FMS-like tyrosine kinase 3 (FLT3) gene occur in approximately 30% of AML cases and are linked to poor prognosis. The discovery of FLT3 inhibitors (FLT3i) has represented a breakthrough in targeted AML therapy; However, resistance to FLT3i remains a significant clinical challenge that limits long-term efficacy. Previously, we identified Sprouty RTK Signaling Antagonist 3 (SPRY3)—a negative regulator of RAS/MAPK signaling—as a key determinant of FLT3i sensitivity. Loss of SPRY3 activated RAS signaling and conferred robust FLT3i resistance in AML. Consistent with these findings, activating NRAS mutations (such as NRASG12C and NRASQ61K) —present in approximately 15% of AML cases—also drive resistance to FLT3i. In our recent studies, we identified ribonucleotide reductase (RNR) as a critical downstream effector upregulated by NRAS-activating mutations (NRASmut) in AML, driving FLT3i resistance. RNR catalyzes the conversion of ribonucleoside diphosphates (NDPs) into deoxyribonucleoside diphosphates (dNDPs), which are subsequently phosphorylated to deoxyribonucleoside triphosphates (dNTPs)—the essential building blocks for DNA replication and repair. We found that NRASmut AML cells exhibit significantly elevated expression of RRM1, RRM2, and RRM2B, the three subunits of RNR, following FLT3i treatment. Mechanistically, RAS activation enhances RNR expression through the RAS-MAPK-E2F1/MYC signaling axis. Inhibition of E2F1 or MYC markedly reduced RRM1 and RRM2 expression, confirming their transcriptional regulation of RNR. Furthermore, knockdown of Dual Specificity Phosphatase 6 (DUSP6)—a negative feedback regulator of RAS/MAPK signaling—further increased RNR activity and sensitized NRASmut AML cells to RNR inhibition. Functionally, both pharmacologic inhibition of RNR using clofarabine and siRNA-mediated knockdown of RNR subunits effectively restored FLT3i sensitivity in NRASmut AML. Combination therapy with gilteritinib and clofarabine in both cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models significantly reduced leukemic burden and prolonged survival compared with either agent alone. In summary, our findings identify RNR as a critical downstream effector of RAS/MAPK signaling and a promising therapeutic target to overcome FLT3i resistance in NRASmut AML. These results provide a strong mechanistic and preclinical rationale for the combined inhibition of FLT3 and RNR as a novel therapeutic strategy to improve outcomes in resistant AML. Citation Format: Zhen Tian, Peng Wang, Stacia Octaviani, Yahui Li, Yun Liao, Xiaolei Liu, Zhaorui Lian, Hong Zheng, Elliot Stieglitz, Catherine C. Smith, Jian Huang. Targeting ribonucleotide reductase to overcome FLT3 inhibitor resistance in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1890.

Abstract Managing treatment toxicity is a major challenge in oncology. Large-scale data on treatment related adverse events (AEs) could help oncologists better understand and predict toxicity. However, most AE datasets are limited to small numbers or restricted types of events due to the labor-intensive manual reporting traditionally required to document AEs. Automated extraction of AEs from the electronic health record (EHR) could enable large-scale analysis but is a challenging task as information about most AEs is in diffuse, unstructured data such as clinical notes.We therefore developed and validated an agentic large language model (LLM) pipeline, AE-Extract, to extract and grade AEs from oncologists’ progress notes. The pipeline is highly sensitive with a recall of >95% for events in the same organ system and a precision of 67% compared to manual annotation. Lower precision relative to recall reflects uncertainty in the attribution of events to treatment effects as well as design choices to prioritize higher recall over precision. To comprehensively characterize AEs during cancer therapy, we subsequently applied AE-Extract to a set of 84,684 progress notes from an academic cancer center. We identified 279,457 total AEs, a mean of 3.3 per note. To identify recurrent patterns of treatment toxicity, we applied non-negative matrix factorization (NMF) to the AE data and identified 85 latent factors that capture patterns of toxicities in the dataset. These latent patterns capture co-occurrence of mechanistically related events such as neuropathy and falls as well as associations in different organ systems without known mechanistic connection such as pruritus and colitis in patients receiving immunotherapy or neuropathy and nail changes in patients receiving cytotoxic chemotherapy. As the latent factors quantify core patterns of AEs, we next studied how they could be used as robust feature sets for analysis of treatment toxicity. First, we found that the latent patterns identified in the first part of a patient’s treatment course can predict development of new AEs as well as worsening severity of existing AEs. We also found these patterns of treatment toxicity correlated with patient demographics and comorbidities: we recapitulated known associations such as the link between gender and cancer induced nausea/vomiting as well as novel relationships such as a link between ischemic heart disease and vestibulocochlear symptoms including tinnitus and hearing impairment.Overall, our work demonstrates a new method for accurate, high-throughput extraction of AEs from clinical notes. We show how comprehensive evaluation of treatment toxicity allows for better characterization of patterns of AEs, identifies robust associations between treatment toxicity and patient characteristics, and may serve as a starting point for building personalized models to predict treatment toxicity. Citation Format: John Lazar, Divneet Mandair, Catherine C. Smith, Travis Zack. Large scale extraction of adverse events by large language models uncovers latent structure of treatment toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2755.

ABSTRACT:Aberrant activation of RAS/MAPK signaling limits the clinical efficacy of several targeted therapies in acute myeloid leukemia (AML). In FMS-like tyrosine kinase-3 (FLT3)-mutant AML, the selection of clones harboring heterogeneous RAS mutations drives resistance to FLT3 inhibitors (FLT3i). RAS activation is also associated with resistance to other AML targeted therapies, such as the B-cell lymphoma 2 inhibitor venetoclax. Despite the critical need to inhibit RAS/MAPK signaling in AML, no targeted therapies have demonstrated a clinical benefit in RAS-driven AML. To address this unmet need, we investigated the preclinical activity of RMC-7977, a multiselective inhibitor of GTP-bound active (RAS[ON]) isoforms of mutant and wild-type RAS in AML models. RMC-7977 exhibited potent antiproliferative and proapoptotic activity across AML cell lines with MAPK-activating signaling mutations. In cell line models with acquired FLT3i resistance because of secondary RAS mutations, treatment with RMC-7977 restored sensitivity to FLT3i. Similarly, RMC-7977 effectively reversed resistance to venetoclax in RAS-addicted cell line models with both RAS wild-type and mutant genetic backgrounds. In murine patient-derived xenograft models of RAS-mutant AML, RMC-7977 was well tolerated and significantly suppressed leukemic burden in combination with gilteritinib or venetoclax. Our findings strongly support clinical investigation of broad-spectrum RAS(ON) inhibition in AML to treat and potentially prevent drug resistance because of activated RAS signaling.

Bulk sequencing of relapsed tumors reveals mutations associated with resistance to cancer therapy but is insufficient to fully assess all causes of relapse. Due to inherent tumor heterogeneity, on-treatment tumor evolution may select for genetically distinct clones or shifts in malignant transcriptional states not resolvable by bulk sequencing. We performed multiomic single cell (SC) DNA/protein and RNA/protein profiling of a clinical trial cohort of acute myeloid leukemia (AML) patients treated on the Phase 1b clinical trial of the BCL2 inhibitor venetoclax and the FLT3 inhibitor gilteritinib (Ven/Gilt) to characterize immunophenotypic, transcriptional, and genetic clonal evolution driving resistance. We found that while Ven/Gilt effectively eliminated FLT3 mutant clones, resistance was associated with RAS activation via multiple mechanisms including selection for RAS mutant clones, non-mutational upregulation of RAS transcriptional programs and a shift to RAS-associated monocytic AML differentiation. In an in vitro model of monocytic differentiation associated with non-mutational RAS transcriptional activation, we demonstrated that RAS pathway inhibition re-sensitized to Ven/Gilt. These data illustrate that convergent resistance pathways in patients can be activated via diverse genetic and non-genetic mechanisms. These results underscore that RAS signaling is central to FLT3 and BCL2 inhibitor resistance, is tightly coupled to AML monocytic differentiation and highlight RAS pathway inhibition as a viable clinical strategy to combat resistance. CT# NCT03625505.

Despite efficacy of FLT3 and BCL2 inhibition in acute myeloid leukemia (AML), relapse limits survival. Mutation status and AML monocytic differentiation are implicated in resistance. On-treatment tumor evolution may select for genetically distinct clones or shifts in differentiation not resolvable by bulk sequencing. We performed multiomic single cell (SC) DNA/protein and RNA/protein profiling of patients treated on a clinical trial of the BCL2 inhibitor venetoclax and the FLT3 inhibitor gilteritinib (Ven/Git) to characterize immunophenotypic, transcriptional, and genetic clonal evolution on therapy. We found that while Ven/Gilt effectively eliminated FLT3 mutant clones, it selected for RAS mutations, RAS pathway activation and RAS-associated monocytic differentiation. In an in vitro model of monocytic differentiation associated with heightened RAS pathway activation, we demonstrated that MEK inhibition re-sensitized to Ven/Gilt. These data indicate RAS signaling is central to FLT3 and BCL2 inhibitor resistance, is tightly coupled to monocytic differentiation and can be overcome by RAS pathway inhibition. ### Competing Interest Statement C.C.S. has provided educational talks for Astellas Pharma, served on advisory boards for Genentech/Abbvie and received research funding from Abbvie. B.C., Y.S. and J.H. are employees of Abbvie. M.S. and H.H. are or were previously employees of Genentech.

Aberrant activation of the RAS/MAPK signaling limits the clinical efficacy of several targeted therapies in acute myeloid leukemia (AML). In FLT3-mutant AML, the selection of clones harboring heterogeneous RAS mutations drives resistance to FLT3 inhibitors (FLT3i). RAS activation is also associated with resistance to other AML targeted therapies, including the BCL2 inhibitor venetoclax. Despite the critical need to inhibit RAS/MAPK signaling in AML, no targeted therapies have demonstrated clinical benefit in RAS-driven AML. To address this unmet need, we investigated the preclinical activity of RMC-7977, a multi-selective inhibitor of GTP-bound active [RAS(ON)] isoforms of mutant and wild-type RAS in AML models. RMC-7977 exhibited potent antiproliferative and pro-apoptotic activity across AML cell lines with MAPK-activating signaling mutations. In cell line models with acquired FLT3i resistance due to secondary RAS mutations, treatment with RMC-7977 restored sensitivity to FLT3i. Similarly, RMC-7977 effectively reversed resistance to venetoclax in RAS-addicted cell line models with both RAS wild-type and mutant genetic backgrounds. In murine patient-derived xenograft models of RAS-mutant AML, RMC-7977 was well tolerated and significantly suppressed leukemic burden in combination with gilteritinib or venetoclax. Our findings strongly support clinical investigation of broad-spectrum RAS(ON) inhibition in AML to treat and potentially prevent drug resistance due to activated RAS signaling.

MOTIVATION:Single-cell DNA sequencing (scDNA-seq) and multi-modal profiling with the addition of cell-surface antibodies (scDAb-seq) have recently provided key insights into cancer heterogeneity. Scaling these technologies across large patient cohorts, however, is cost and time prohibitive. Multiplexing, in which cells from unique patients are pooled into a single experiment, offers a possible solution. While multiplexing methods exist for scRNAseq, accurate demultiplexing in scDNAseq remains an unmet need. RESULTS:Here, we introduce SNACS: single-nucleotide polymorphism and antibody-based cell sorting. SNACS relies on a combination of patient-level cell-surface identifiers and natural variation in genetic polymorphisms to demultiplex scDNAseq data. We demonstrated the performance of SNACS on a dataset consisting of multi-sample experiments from patients with leukemia where we knew truth from single-sample experiments from the same patients. Using SNACS, accuracy ranged from 0.948 to 0.991 versus 0.552 to 0.934 using demultiplexing methods from the single-cell literature. AVAILABILITY AND IMPLEMENTATION:SNACS is available at  https://github.com/olshena/SNACS.

Motivation Single-cell DNA sequencing (scDNA-seq) and multi-modal profiling with the addition of cell-surface antibodies (scDAb-seq) have recently provided key insights into cancer heterogeneity. Scaling these technologies across large patient cohorts, however, is cost and time prohibitive. Multiplexing, in which cells from unique patients are pooled into a single experiment, offers a possible solution. While multiplexing methods exist for scRNAseq, accurate demultiplexing in scDNAseq remains an unmet needResults Here, we introduce SNACS: single-nucleotide polymorphism and antibody-based cell sorting. SNACS relies on a combination of patient-level cell-surface identifiers and natural variation in genetic polymorphisms to demultiplex scDNAseq data. We demonstrated the performance of SNACS on a dataset consisting of multi-sample experiments from patients with leukemia where we knew truth from single-sample experiments from the same patients. Using SNACS, accuracy ranged from 0.948 to 0.991 versus 0.552 to 0.934 using demultiplexing methods from the single-cell literature.Availability and implementation SNACS is available at https://github.com/olshena/SNACS.

Mutations of isocitrate dehydrogenase (IDH) are recurrent in newly diagnosed (ND) acute myeloid leukemia (AML) and the prevalence increases with age. The prognostic impact of IDH mutations in AML remains controversial. IDH inhibitors generally have a favorable side effect profile, making them an attractive option for older patients. This retrospective analysis aimed to describe the prevalence and prognostic impact of IDH mutations in a large cohort of ND AML patients aged ≥60 years enrolled in the Beat AML clinical trial. A total of 1,023 patients were included. IDH mutations were detected in 28% of the patients, including 9.7% IDH1mut, 18.9% IDH2mut, and 1.0% had a mutation in IDH1 and IDH2. IDH frequently co-occurred with DNMT3A (38%), NPM1 (35%), and SRSF2 (34%). In patients treated with intensive chemotherapy, IDH mutations were not prognostic for overall survival (OS) (p=0.76), while OS was longer for patients with IDH2mut compared to IDHwt in patients treated with hypomethylating agent (HMA)-based therapy (median OS of 18.5 vs 10.2 months, p

Normal and oncogenic Ras proteins are functionally dependent on one or more lipid modifications 1,2 . Whereas K-Ras4b farnesylation is sufficient for stable association with the plasma membrane, farnesylated H-Ras, K-Ras4a, and N-Ras traffic to the Golgi where they must undergo palmitoylation before regulated translocation to cell membranes. N-Ras palmitoylation by the DHHC family of palmitoyl acyl transferases (PATs) and depalmitoylation by ABHD17 serine hydrolases is a dynamic process that is essential for the growth of acute myeloid leukemias (AMLs) harboring oncogenic NRAS mutations 3-6 . Here, we have tested whether co-targeting ABHD17 enzymes and Ras signal output would cooperatively inhibit the proliferation and survival of NRAS -mutant AMLs while sparing normal tissues that retain K-Ras4b function. We show that ABD778, a potent and selective ABHD17 inhibitor with in vivo activity, selectively reduces the growth of NRAS -mutant AML cells in vitro and is synergistic with the allosteric MEK inhibitor PD0325901 (PD901) 7,8 . Similarly, ABD778 and PD901 significantly extended the survival of recipient mice transplanted with three independent primary mouse AMLs harboring an oncogenic Nras G12D driver mutation. Resistant leukemias that emerged during continuous drug treatment acquired by-pass mutations that confer adaptive drug resistance and increase mitogen activated protein kinase (MAPK) signal output. ABD778 augmented the anti-leukemia activity of the pan-PI3 kinase inhibitor pictilisib 9 , the K/N-Ras G12C inhibitor sotorasib 10 , and the FLT3 inhibitor gilteritinib 11 . Co-treatment with ABD778 and gilteritinib restored drug sensitivity in a patient-derived xenograft model of adaptive resistance to FLT3 inhibition. These data validate the palmitoylation cycle as a promising therapeutic target in AML and support exploring it in other NRAS -mutant cancers.

Mixed phenotype acute leukemia (MPAL) is a leukemia whose biologic drivers are poorly understood, therapeutic strategy remains unclear, and prognosis is poor. We performed multiomic single cell (SC) profiling of 14 newly diagnosed adult MPAL patients to characterize the immunophenotypic, genetic, and transcriptional landscapes of MPAL. We show that neither genetic profile nor transcriptome reliably correlate with specific MPAL immunophenotypes. However, progressive acquisition of mutations is associated with increased expression of immunophenotypic markers of immaturity. Using SC transcriptional profiling, we find that MPAL blasts express a stem cell-like transcriptional profile distinct from other acute leukemias and indicative of high differentiation potential. Further, patients with the highest differentiation potential demonstrated inferior survival in our dataset. A gene set score, MPAL95, derived from genes highly enriched in this cohort, is applicable to bulk RNA sequencing data and was predictive of survival in an independent patient cohort, suggesting utility for clinical risk stratification.