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.

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.

Overall survival based on (A) NPM1 mutation status, (B) FLT3-ITD mutation status within NPM1 mutated cases only, (C) IDH1 mutation status, and (D) IDH2 mutation status. Reported p-values utilized the cox model Wald test.

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

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

Surface expression of CD14, CD64, and CD11b in cases by genetic mutation compared to a wild-type reference cohort within the subset of patients with centralized quantitative MFC performed (n = 144). * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. ref: reference.

Overview of immunophenotypic expression profiling. (A) Flow cytometry was performed on diagnostic bone marrow specimens and fluorescence measurements, forward light scatter (FSC) and right-angle light scatter (SSC) characteristics were collected for 200,000 events. Leukemic populations were identified by CD45 v SSC gating, and, in a small subset of cases with markedly hypogranular myeloids, by CD34 gate (these cases all contained an entirely CD34 positive blast population). Final gating for every case was reviewed by a pathologist. (B) For cells identified in the leukemia gate, the mean intensity for each parameter (represented by a black dot) was computed.

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

ABSTRAT:MDM2 inhibitors are promising therapeutics for acute myeloid leukemia (AML) with wild-type TP53. Through an integrated analysis of functional genomic data from primary patient samples, we found that an MDM2 inhibitor, idasanutlin, like venetoclax, is ineffective against monocytic leukemia (French-American-British [FAB] subtype M4/M5). To dissect the underlying resistance mechanisms, we explored both intrinsic and extrinsic factors. We found that monocytic leukemia cells express elevated levels of CEBPB, which promote monocytic differentiation, suppress CASP3 and CASP6, and upregulate MCL1, BCL2A1, and the interleukin (IL-1)/tumor necrosis factor alpha (TNF-α)/NF-κB pathway members, thereby conferring drug resistance to a broad range of MDM2 inhibitors, BH3 mimetics, and venetoclax combinations. In addition, aberrant monocytes in M4/M5 leukemia produce elevated levels of IL-1 and TNF-α, which promote monocytic differentiation and upregulate inflammatory cytokines and receptors, thereby extrinsically protecting leukemia blasts from venetoclax and MDM2 inhibition. Interestingly, IL-1β and TNF-α only increase CEBPB levels and protect M4/M5 cells from these drugs but not M0/M1 leukemia cells. Treatment with venetoclax and idasanutlin induces compensatory upregulation of CEBPB and the IL-1/TNF-α/NF-κB pathway independent of the FAB subtype, indicating drug-induced compensatory protection mechanisms. The combination of venetoclax or idasanutlin with inhibitors that block the IL-1/TNF-α pathway demonstrates synergistic cytotoxicity in M4/M5 AML. As such, we uncovered a targetable positive feedback loop that involves CEBPB, IL-1/TNF-α, and monocyte differentiation in M4/M5 leukemia and promotes both intrinsic and extrinsic drug resistance and drug-induced protection against venetoclax and MDM2 inhibitors.

Supplementary Figure 3: Vβ monoclonal antibodies induce antibody dependent cellular cytotoxicity of target cells.

Resistance to venetoclax (VEN)-based therapy in acute myeloid leukemia (AML) includes genetic (i.e., mutations in N/KRAS, FLT3-ITD, TP53) and phenotypic (i.e., monocytic differentiation) features. Whether monocytic differentiation contributes to clinical VEN resistance secondary to a genetic bias remains unknown. This multimodal, multicenter, international analysis, inclusive of 678 patients, comprehensively characterized the prognostic role of monocytic differentiation in patients with AML treated with hypomethylating agents combined with VEN. AML genetics and monocytic differentiation (HR = 1.89; 95% confidence interval, 1.35-2.66; P < 0.001) in NPM1 wild-type cases correlated with an increased risk of death, clustering of centralized quantitative multiparameter flow cytometry data, evaluation of RNA sequencing-derived AML maturation stage, and single-cell proteogenomics linked driver mutations with AML phenotype and antiapoptotic gene expression. This comprehensive analysis of AML genetics, phenotype, and antiapoptotic protein expression highlights the complementary role these factors impart following VEN-based therapy. SIGNIFICANCE:AML with monocytic differentiation often occurs in the context of co-occurring mutations within signaling pathways. In certain AML subgroups (such as NPM1 wild-type and signaling pathway gene-mutated), a monocytic phenotype is associated with decreased overall survival following VEN-based therapy. See related commentary by Renders, p. 403.

Patient and disease characteristics for Beat AML RNAseq cohort (N = 228)

Overall survival in patients classified as monocytic vs. non-monocytic in (A) patients classified as mPRS higher-benefit (wild-type for FLT3, N/KRAS and TP53), (B) with mutated IDH1/2 and wild-type for N/KRAS, PTPN11, FLT3-ITD, and TP53, and (C) wild-type for NPM1, FLT3-ITD, IDH1/2, K/NRAS, PTPN11, and TP53. Reported p-values utilized the cox model Wald test.

Venetoclax (ven) combined with azacytadine is a widely used therapy for acute myeloid leukemia (AML). However, most patients develop resistance. To identify more effective combinations, we analyze 302 AML patient samples and find ven plus palbociclib (ven+palbo), a cyclin dependent kinase (CDK)4/6 inhibitor, to be highly effective. Ven+palbo shows synergistic activity in AML cell lines and patient-derived xenograft mouse models. Patient samples exhibiting a synergistic response to ven+palbo show downregulation of genes involved in protein synthesis. Genome-wide (CRISPR) screening shows that loss of translational genes uniquely confers sensitivity to ven but not to ven+palbo. AML cells exposed to ven exhibit an adaptive increase of protein synthesis that is overcome by ven+palbo through regulation of translational machinery. Additionally, ven+palbo mitigates resistance mechanisms observed with single-agent ven (BAX loss) and palbo (RB1 loss). Finally, we identify the loss of IKZF1 as a mechanism of resistance to ven+palbo and show that targeting AXL is effective in IKZF1-mutated AML.

RNA expression of alternative anti-apoptotic proteins based on RNA-seq defined cell differentiation state. Samples from the top quartile of each state are displayed below.

Overall survival within the US cohort (n = 279) based on the presence of signaling pathway mutations including (A) NRAS, (B) KRAS, (C) PTPN11, (D) BRAF, (E) NF1, and (F) WT1. Reported p-values utilized the cox model Wald test.

Supplementary Table S1. (A) List of published datasets used to compile the distribution of TCRVB Gene Usage across T-cell NHL. (B) TCR Vβ usage in 314 patients with T-cell NHL by disease type. Note that both the IMGT and Wei et al nomenclature are listed.

The emergence of transcriptional signatures that define cell types and pathways has made it possible to guide cancer therapy selection through gene expression profiling. We developed a rapid qPCR-based platform to profile cell state, stemness, and BCL2 family gene expression as a companion diagnostic test for acute myeloid leukemia (AML). We validated the stability and utility of the signatures across multiple measurement platforms and using patient samples from two centers. Integrating these signatures with clinical features enables an expedient means to predict the likelihood of patient responses to two standard-of-care therapies: intensive chemotherapy and hypomethylating agent plus venetoclax (HMA+Ven). For patients treated with HMA+Ven, expression levels of the promonocyte-like signature and BCL2 add predictive value for response and overall survival in multivariable models that include genetic features. The incorporation of the rapid profiler into the prospective evaluation of newly diagnosed AML patients may enhance treatment stratification and improve outcomes.

Overall survival in the subgroup of patients not receiving allogeneic cell transplantation (HCT) based on (A) monocytic vs. non-monocytic differentiation, (B) monocytic differentiation and NPM1 mutation status, (C) in the subgroup of patients with RAS pathway and FLT3-ITD mutations (K/NRAS, PTPN11, FLT3-ITD) and wild-type IDH1/2, NPM1, and TP53; (D) within the patient subgroup classified as mPRS higher-benefit (wild-type for FLT3, N/KRAS and TP53). Reported p-values utilized the cox model Wald test.