Functional small-molecule screening of primary acute myeloid leukemia (AML) samples identified ex vivo synergy between the JAK1/2 inhibitor, ruxolitinib (Rux), and venetoclax (Ven) in newly diagnosed and relapsed/refractory (R/R) AML. This motivated a phase 1 multicenter trial to evaluate Rux + Ven in 30 patients with R/R AML. Patients had median age of 69 years and were heavily pretreated (40% with ≥3 previous lines, 43% with previous Ven failure). Rux (30 mg twice daily) and Ven (400 mg once daily) were well tolerated without dose-limiting toxicities. Median duration of therapy was 55 days. Over 2 cycles of Rux + Ven, clinical response rate was 20% and composite complete remission (CR) rate was 10%. Median survival was 3.7 months, with 23% alive at 1 year. There were 2 exceptional responders, including a patient who remained in CR/CR with incomplete hematologic recovery for nearly 4 years after hypomethylating agent + Ven failure. CD56 expression on blasts was significantly associated with lack of clinical response (odds ratio, 0.09; P = .039) and shorter survival (hazard ratio, 2.35; P = .048). In a subset of patients (n = 8), cytometry by time of flight comparison of primary cells at pretreatment vs day 8 showed upregulation of pCREB and downregulation of CD11b in nonresponders. In summary, the novel combination of Rux + Ven is safe in adult patients with R/R AML. Our study identified cellular and molecular biomarkers, notably CD56, that predict resistance to Rux + Ven, but further work is needed to understand and validate their effect in AML. This trial was registered at www.clinicaltrials.gov as #NCT03874052.

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.

Azacitidine (Aza) plus venetoclax (Ven) is standard treatment for older/unfit patients with newly diagnosed (ND) acute myeloid leukemia (AML). The approved 28-day (D) Ven schedule is associated with prolonged cytopenias, causing frequent dose reductions and cycle delays. Retrospective studies show similar efficacy and reduced toxicity with abbreviated Ven dosing, but prospective data is lacking. We conducted OPTI-AML(NCT03013998), a prospective randomized phase 2 trial comparing 28D Ven (AV28) versus 14D (AV14) with Aza (75mg/m²x7D) for C1-2 in genomically agnostic ND-AML patients ≥60 years. The primary endpoint was complete remission (CR) rate achieved at any time with two cycles of therapy. Between 2023-2025, 169 patients received AV28 (n=83) or AV14 (n=86). CR across two cycles was 49.4% (AV28) versus 43% (AV14); difference of 6.4% [90%CI:-6.1% to 19.0%], not meeting non-inferiority criteria. Patients with NPM1/ IDH2 mutations had higher CR rates with AV28 (60.9% vs. 33.3%), while CR rates were equivalent (45%) for other subgroups. Composite CR rates were 80.7% (AV28) versus 68.6% (AV14) and MRD negativity was similar (77.6% vs. 76.5%). Although AV28 had more frequent treatment interruptions, count recovery after C2, grade ≥3 adverse events and early mortality were similar. In conclusion, the study did not demonstrate non-inferiority of AV14 compared with AV28 during C1-2 in an unselected ND-AML cohort. However, as the confidence interval for the difference covered 0, CR rate for AV28 was not significantly different than AV14. Certain subgroups may benefit from prolonged Ven exposure, but these findings require validation in larger studies, especially as triplet regimens evolve.

While the FLT3 inhibitor gilteritinib is initially effective in patients with FLT3-mutated acute myeloid leukemia (AML), patients invariably relapse within months of treatment. Gilteritinib resistance is commonly driven by the emergence of NRAS mutations and a shift towards a more monocytic cell state. We hypothesized that directly targeting and depleting NRAS protein would reverse these adaptive differentiation changes and restore therapeutic sensitivity. To test this, we utilized a mutation-agnostic antisense oligonucleotide (ASO) to selectively knock down NRAS expression across gilteritinib-resistant AML cell lines, in vivo cell-line-derived xenografts, and primary patient samples. NRAS ASO successfully resensitized gilteritinib resistant cells with multiple distinct NRAS mutations, displaying superior efficacy compared to downstream MEK inhibition. This therapeutic efficacy was independent of NRAS mutant variant allele frequency, suggesting that wild-type NRAS may also contribute to resistance. Comprehensive multi-omic profiling (transcriptomics, proteomics, phosphoproteomics) revealed that NRAS knockdown consistently reversed monocytic phenotype, shifting cells back toward a more primitive cell state. Monocytic differentiation and NRAS mutations are established drivers of resistance to diverse targeted regimens in AML, including FLT3, IDH, and BCL2 inhibitors, so we also tested venetoclax resistant primary cells with NRAS mutations. Resistant cells were resensitized to venetoclax after NRAS knockdown, suggesting that NRAS knockdown may be more broadly applicable in overcoming monocytic cell state and drug resistance in AML.

6598 Background: Treatment with tyrosine kinase inhibitors (TKIs) enables patients with chronic myeloid leukemia (CML) to achieve durable remission and a normal life expectancy. Despite this success, chronic TKI therapy is associated with cumulative toxicities. Although TKI dose de-escalation can be implemented to mitigate adverse events, its impact on the attainment and durability of response milestones is not completely defined. This study explored the impact of dose de-escalation of TKIs on clinical response milestones and treatment-free remission (TFR) outcomes. Methods: We retrospectively analyzed 407 adult CML patients treated with TKIs at Oregon Health & Science University. Outcomes of interest were TFR and achievement of undetectable BCR::ABL1 transcripts. Odds ratios (OR) or Cox proportional hazards regression hazard ratios (HR) were used to analyze covariates and predictors. Results: Overall, the median age at diagnosis was 48 years (range: 18-91) and 1 st -line therapies included: imatinib (n=252; 62%), dasatinib (n=95; 23%), nilotinib (n=42; 10%), and bosutinib (n=10; 2%); the remaining 2% of patients were treated with 3 rd generation or later TKIs. Median follow-up since diagnosis was 11.3 years (0.2-35.8); 189 patients (46%) remained on their initial TKI, while 218 patients (54%) received ≥2 TKIs. Only 13 patients (3.2%) received a bone marrow transplant. With respect to the primary outcome of TFR, 133 patients attempted TFR and were further analyzed with respect to de-escalation of dose. Fifty-two patients (39%) remained on standard dose while 81 patients (61%) underwent dose de-escalation prior to the 1 st TFR attempt. TFR rates were not significantly different between patients receiving standard dose vs de-escalated dose TKI (HR: 1.07 (95% CI: 0.59-1.96); p=0.818) at either 1 year (77% vs 74%) or 2 years (69% vs 69%). At the last follow-up, 89 patients remained in TFR, 62% of which had undergone dose de-escalation. Among the remaining 274 patients who have yet to attempt TFR, 162 (59%) continued treatment with their initial TKI: 98 (60%) on imatinib, 39 (24%) on dasatinib, 17 (10%) on nilotinib, 5 (3%) on bosutinib, 2 (1%) on ponatinib, 1 (1%) on asciminib. Among these patients, 56 (34%) had dose reduction, 9 (16%) of whom achieved undetectable disease. For the 106 patients on standard doses, only 5 (5%) had undetectable disease (OR: 3.83 (95% CI: 1.08-15.40); p= 0.020).More importantly, dose de-escalation led to significant reduction of adverse events. Conclusions: Our results show that patients who underwent dose de-escalation did not have significant differences in TFR duration and rate and are more likely to achieve undetectable diseases when compared with patients on standard doses. These findings support rational dose de-escalation as a strategy that preserves achievement of clinical outcomes while reducing adverse events.

Abstract Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL1 fusion gene in chronic myeloid leukemia (CML) have improved patient prognosis. While newer drugs have made inroads against BCR-ABL1-dependent TKI resistance, patients who develop resistance through pathways independent of BCR-ABL1 feature heterogeneous, poorly characterized molecular mechanisms and present a challenge for application of combination targeted therapy. To identify genes contributing to BCR-ABL1-independent resistance, we performed CRISPR/Cas9 genome-wide screens using Ba/F3 BCR-ABL1 cells cultured in the presence of DMSO, imatinib, and asciminib. After 10 days, cells were harvested and analyzed for gene-level enrichment/depletion of sgRNAs. Among candidate resistance genes (whose knockdown was enriched following TKI treatment), independent sgRNA guides for five genes were induced by lentiviral CRISPR/Cas9 knockout in Ba/F3 BCR-ABL1 cell line models for validation studies. Cell lines were evaluated for in vitro sensitivity to a panel of approved ABL1 TKIs and profiled against an expanded inhibitor panel spanning a range of drug targets. Expression levels of candidate resistance genes were compared by RNAseq in primary specimens from CML patients. CRISPR screening revealed varying subsets of genes enriched in TKI-treated cultures. Five candidate genes were selected for validation studies: Chic2, Stub1 and Pten from the imatinib-treated cells and Fbxo3 and Ptar1 from the asciminib-treated cells. In Ba/F3 BCR-ABL1 cell line models, knockout of each of these genes resulted in varying degrees of reduced sensitivity to a panel of ABL1 TKIs (2-45-fold increase in IC50 compared to wild-type cells). For example, knockout of Pten and Chic2 demonstrated increased IC50 values for imatinib of 12,714 and 914 nM, respectively, compared to 277 nM for wild-type cells. Profiling of cell lines using an expanded drug panel revealed differential sensitivities to inhibitors targeting multiple pathways. For example, Chic2, Stub1 and Fbxo3 knockout showed greater sensitivity (relative to wild-type cells) to inhibitors of WNT, proteasome, histone deacetylase, bromodomain, and/or nucleoside analog pathways. Lastly, analysis of RNASeq data highlighted differences in expression levels in patient samples. For example, expression levels of both Ptar1 and Pten were reduced in patients with BCR-ABL1-independent resistance to imatinib relative to newly diagnosed patients, and expression of Fbxo3 and Chic2 were decreased with disease progression (blast vs chronic phase). Taken together, our results identify genes implicated in tumor suppression (Pten), ubiquitination (Stub1, Fbxo3, Chic2), and post-translational modification (Ptar1) with contributing roles for BCR-ABL1-independent TKI resistance and map these to potential actionable pathways amenable to novel combination targeted therapy approaches. Citation Format: Mark Pusung, Christopher A. Eide, Jessica Gibbs, Daniel Bottomly, Haijiao Zhang, Brian J. Druker. Targeting BCR-ABL1-independent mechanisms of resistance in chronic 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 6499.

SPI1 knockdown does not disrupt dual FLT3/LSD1 inhibition synergy.

Regulon Enrichment Analysis of Primary AML Blast Bulk RNA-seq from 681 Patient Samples

Supplementary Figure S1. Immunophenotype of Patient’s T-LGL cells.

Efficacy of dual FLT3/LSD1 inhibition in MOLM13 cells.

Supplementary Figure S2: The binding specificity of αVβ monoclonal antibodies.

Transcription Factor Enrichment Analysis of Primary AML Bulk RNA-Seq (24 Hours After Drug Treatment)

Supplementary Materials and Methods

PURPOSE:Azacitidine and venetoclax is a standard frontline treatment regimen for newly diagnosed older adults with AML; however, long-term outcomes remain poor. Revumenib is an oral menin inhibitor with clinical activity in AML patients with nucleophosmin-1 mutation (NPM1m) or lysine methyltransferase 2A rearrangements (KMT2Ar). METHODS:We conducted a phase I dose-escalation and expansion study of azacitidine, venetoclax, and revumenib at two dose levels (113 mg or 163 mg orally every 12 hours in combination with strong cytochrome P450 inhibitor azoles) in patients aged 60 years and older newly diagnosed with AML with NPM1m or KMT2Ar (ClinicalTrials.gov identifier: NCT03013998). RESULTS:Overall, 43 patients were enrolled and treated. There was no maximal tolerated dose identified. Differentiation syndrome was present in eight (19%) patients and QTc Fridericia prolongation was present in 19 (44%) patients, and neither required permanent discontinuation of revumenib. The overall response rate with an intention-to-treat population was 88.4% (95% CI, 74.9 to 96.1; NPM1m: 85.3%; KMT2Ar: 100%), the rate of composite complete remission (complete remission [CR] + CR with partial or incomplete hematologic recovery) was 81.4% (95% CI, 66.6 to 91.6; NPM1m: 79.4%; KMT2Ar: 88.9%), and the rate of CR was 67.4% (95% CI, 51.5 to 80.9; NPM1m: 65%; KMT2Ar: 78%). No patient had refractory disease after 1-2 cycles of treatment. The median time to first response was 28 days, and 84% of responders achieved remission within the first cycle. All 37 patients evaluated had no evidence of measurable residual disease by a centralized flow cytometry assay. CONCLUSION:In older adults newly diagnosed with NPM1m or KMT2Ar AML, the combination of azacitidine, venetoclax, and revumenib was able to be safely administered with high rates of CR and clinical activity.

Supplementary Figure 5. αVβ monoclonal antibodies induce target T-cell activation, characterized by down modulation of the TCR complex

Excess over Bliss of Primary AML Blasts from 72 Patient Samples

Correlation of Regulon Enrichment Signatures with MYC Gene Expression in Primary AML