Biomedical machine learning (ML) models raise critical concerns about embedded assumptions influencing clinical decision-making, necessitating robust documentation frameworks for datasets that are shared via external repositories. Fairness-aware algorithm effectiveness hinges on users' prior awareness of specific issues in the data - information such as data collection methodology, provenance and quality. Current ML-focused documentation approaches impose impractical burdens on data generators and conflate data/model accountability. This is problematic for resource datasets not explicitly created for ML applications. This study addresses these gaps through a two-step process: First, we derived consensus documentation fields by mapping elements across four key templates. Second, we surveyed biomedical stakeholders across four roles (clinicians, bench scientists, data manager and computationalists) to assess field importance and relevance. This revealed important role-dependent prioritization differences, motivating the development of the Biomedical Data Manifest - a modular template employing persona-specific field presentation reducing generator burden while ensuring end-users receive role-relevant information. The Biomedical Data Manifest improves transparency for datasets deposited in public or controlled-access repositories and bias mitigation across ML applications.

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

Efficacy of dual FLT3/LSD1 inhibition in MOLM13 cells.

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

Frontline use of the BCL2 inhibitor, venetoclax, for acute myeloid leukemia (AML) has resulted in broad improvements in patients' outcomes. A major remaining challenge is the development of venetoclax resistance, frequently driven by compensatory transcriptional programs that promote cell survival and differentiation. These changes reduce dependence on BCL2 in favor of alternative anti-apoptotic BCL2 family members such as MCL1 or BCL2L1 (BCL-XL). Using CRISPR-based genome-wide perturbation screens, we investigated the genetic dependencies of venetoclax and the BCL2/BCL2L1 dual inhibitor AZD4320. We identified the N6-methyladenosine (m6A) writer RBM15, and the nucleosome remodeling and deacetylase (NuRD) complex interactor ZMYND8 as novel mediators of resistance to both venetoclax and AZD4320. Loss of RBM15 or ZMYND8 induced drug resistance, concurrent with alterations in BCL2 family expression and monocytic differentiation. Accordingly, in AML patients' samples we found reduced expression of the respective m6A or NuRD complexes was significantly associated with monocytic differentiation and ex vivo resistance to the same drugs. These findings provide critical insights into previously undescribed mechanisms of BCL2 family inhibitor resistance in AML.

Supplementary Materials and Methods

Excess over Bliss of Primary AML Blasts from 72 Patient Samples

Given the highly aggressive and heterogeneous nature of metastatic triple-negative breast cancer, molecular subtypes have been evaluated for their utility in patient stratification and therapeutic selection. Leveraging both our unique longitudinal multimodal analysis of serial tumor biopsies, as well as existing public reference cohorts, we refined clinically relevant molecular subtypes through de-novo network-based approaches. A plasma/B-cell related co-expression module emerged as a robust predictor of clinical response. Refinements of this module were significantly associated with pathological complete response and survival in the CALGB and METABRIC cohorts, as well as dramatically improving the call rate in a CLIA setting. We explored patient-specific networks to monitor individual adaptive responses to therapy, allowing for dynamic adjustments in treatment strategies. Our work supports the shift from traditional molecular subtyping towards a more integrated view that includes the tumor microenvironment and immune landscape in a network-based context.

Drug synergy between FLT3 and LSD1 inhibitors in FLT3-ITD and FLT3-wildtype cell lines.

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

Super-Enhancer-Type Analysis of STAT5 ChIP-Seq Peaks (24 Hours After Drug Treatment).

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.

BackgroundAcute myeloid leukemia (AML) is characterized by a complex interplay between genomic alterations, aberrant hematopoiesis, and immune evasion. The aryl hydrocarbon receptor (AHR) pathway is a critical player in this phenomenon determining the fate of stem cell differentiation as well as dictating immune cell development and function. Despite this critical connection, little is known about how AHR regulates the immune microenvironment in AML.MethodsWe performed a retrospective study examining the pre-treatment effect of immune cell numbers (T and NK cells) in the bone marrow and their impact on overall survival in AML patients undergoing 7+3 induction chemotherapy. Utilizing flow cytometry and both bulk and single-cell RNA sequencing of AML patient samples, we characterized the immune signature of blast cells and the influence of AHR on the immune microenvironment. Lastly, we performed functional studies to determine impact of pharmacological and genomic AHR inhibition on NK cell function.ResultsHigher bone marrow NK cell percentage in ND-AML correlated with poorer OS and expression of HLA-E on leukemic blasts. AHR upregulation was associated with HLA-E expression on blasts and an innate immune resistant signature defined by upregulation of key cytokine pathways, interferon gamma (IFN-g) pathway, and MHC class I/II as well as impaired NK cell profiles. High AHR expression in AML was associated with monocytic maturation and discrepant MHC class I/II profiles. Pre-treatment of blasts with an AHR inhibitor (AHRi) prior to NK cell killing assay downregulated key checkpoint molecules, including HLA-E, and key IFN-g signaling transcription factors (STAT1, IRF1) and led to enhanced NK cell killing among multiple FAB subsets in AML.ConclusionThe data support targeting the AHR pathway as a dual tumor intrinsic and immune targeting therapeutic strategy for AML, particularly in combination with NK cellular therapy.

GFI1 knockdown weakens the effects of dual FLT3/LSD1 inhibition.

Even though head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer worldwide, there are only two PD-1 targeted immunotherapies (pembrolizumab and nivolumab) and one tumor intrinsic EGFR targeted therapy (cetuximab) that are FDA approved for treatment of HNSCC. Taking advantage of a high throughput inhibitor assay and computational tools originally showing success in leukemia, we designed and employed HNSCC-specific inhibitor panels that capture the diversity of aberrational pathways in HNSCC to test viable cells derived from patients' HNSCC tumors. This provides a functional context to the multi-omic readouts conducted on these samples (mutations, protein expression and copy number alterations). In addition to generating these deeply characterized functional genomics datasets, we also developed additional visual analytics that have the potential to provide greater insight into HNSCC drug response patterns and potentially aid precision oncology tumor boards in evaluation and assessment of effective targeted therapeutic agents.

Log2 Normalized Counts of Differentially Expressed Genes from MOLM13 Bulk RNA-seq (24 Hours After Drug Treatment)

Epigenetic impact of dual FLT3/LSD1 inhibition.

Efficacy of dual FLT3/LSD1 inhibition in primary AML samples.