ADAM15 drives venetoclax resistance via HCK–AKT–FOXO3A signaling in AML

ADAM15 drives venetoclax resistance via HCK–AKT–FOXO3A signaling in AML | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article ADAM15 drives venetoclax resistance via HCK–AKT–FOXO3A signaling in AML Peilong Lai, Kaifan Liu, Lingji Zeng, Maozhi Xiao, Suxia Geng, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9498017/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Venetoclax-based regimens have improved outcomes in acute myeloid leukemia (AML), but resistance frequently emerges and limits durable benefit. The upstream signaling mechanisms linking therapeutic stress to apoptotic evasion remain poorly defined. Here, we identify ADAM15 as a previously unrecognized regulator of venetoclax resistance in AML. Integrative multi-omics analyses across experimental models and clinical cohorts reveal that ADAM15 is consistently upregulated in venetoclax refractory AML and is associated with diminished drug sensitivity and adverse clinical outcomes. Ectopic expression of ADAM15 in parental sensitive cells confer venetoclax resistance, while genetic depletion of ADAM15 in resistant counterparts restores drug sensitivity and enhances mitochondrial apoptotic priming in vitro, and suppresses leukemia progression in resistant xenograft models. Mechanistically, ADAM15 engages HCK under venetoclax exposure to activate an AKT-FOXO3A survival program, thereby sustaining resistance. Collectively, these findings establish ADAM15 as a convergent and therapeutically actionable vulnerability in venetoclax-refractory AML and provide a translational rationale for targeting this axis to overcome resistance. Health sciences/Medical research/Translational research Health sciences/Diseases/Haematological diseases/Haematological cancer/Leukaemia/Acute myeloid leukaemia Health sciences/Medical research/Preclinical research Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Acute myeloid leukemia (AML) remains a leading cause of leukemia-related mortality in adults. The BCL-2 inhibitor venetoclax (VEN), combined with hypomethylating agents or low-dose chemotherapy, has substantially improved initial response rates in treatment-ineligible patients; however, responses are frequently transient and heterogeneous 1 , 2 , and median overall survival remains only 9–14 months 3 – 5 . The near-universal emergence of resistance is therefore the principal barrier to durable clinical benefit. At the mechanistic level, VEN resistance converges on mitochondrial apoptotic rewiring: resistant AML cells reduce BCL-2 dependence and shift survival reliance toward MCL-1 or BCL-XL 6,7 , reflected functionally by diminished mitochondrial priming and impaired apoptosis induction 8 , 9 . While these downstream adaptations are well characterized, the upstream signaling mechanisms that dynamically orchestrate them under therapeutic stress remain poorly defined, limiting the development of rational combination strategies. A Disintegrin And Metalloproteinase 15 (ADAM15) is a multifunctional transmembrane protein that acts predominantly as a signaling scaffold to promote tumor progression and therapeutic resistance. In solid tumors, ADAM15 drives therapeutic resistance by activating EGFR- and Src family kinase-dependent pro-survival pathways and recruiting kinase-active signaling complexes under stress 10 , 11 . Despite these observations, its role in hematologic malignancies 12 — and specifically in AML therapy resistance — remains largely unexplored. Notably, AKT–FOXO3A signaling, a candidate effector axis downstream of scaffold-mediated kinase activation, is activated in adverse-risk AML and associated with inferior responses to VEN-based regimens 13 – 16 , yet whether this axis is engaged in response to VEN treatment and how it interfaces with mitochondrial apoptotic rewiring remain unknown. Integrating multi-omics data across experimental models and clinical cohorts, we identify ADAM15 as a convergent driver of VEN resistance that engages HCK to activate AKT–FOXO3A signaling and suppress mitochondrial apoptotic priming. Genetic and pharmacologic disruption of this axis restores VEN sensitivity in vitro and in vivo, defining the ADAM15–HCK–AKT–FOXO3A pathway as a therapeutically actionable vulnerability for overcoming BCL-2 inhibitor resistance in AML. Results Venetoclax resistance in AML is driven by phenotypic diversification and anti-apoptotic rewiring Sensitivity profiling across a panel of human AML cell lines identified MOLM-13, OCI-AML-2, MV4-11, and KG1α as VEN-sensitive, and OCI-AML-3 and THP-1 as intrinsically resistant (Supplementary Fig. S1 A). Stepwise dose escalation in three sensitive lines generated resistant derivatives (MOLM-13/R, OCI-AML-2/R, MV4-11/R) with > 10-fold increases in IC₅₀ (Fig. 1 A, Table 1 , Supplementary Fig. S1 B). Table 1 IC50 values and fold changes of Venetoclax in parental and resistant AML cell lines. Cell Line Parental IC50 (µM) [95% CI] Resistant IC50 (µM) [95% CI] Fold Change MOLM-13 0.29 [0.26–0.34] 6.59 [5.44–7.95] 22.39 OCI-AML-2 0.04 [0.036–0.045] 14.02 [11.00–18.15] 345.24 MV4-11 0.03 [0.026–0.032] 2.28 [1.72–3.07] 79.19 VEN-resistant cells displayed markedly attenuated apoptotic responses to ABT-199 (Fig. 1 B, Supplementary Fig. S1 C). Cell-cycle analysis revealed a divergent response to drug exposure: parental cells underwent S-phase reduction and sub-G0 accumulation, whereas resistant cells evaded cell-cycle restraint and maintained S-phase progression (Fig. 1 C). These findings suggest that VEN resistance is associated with a fundamentally altered proliferative response to drug exposure. In parallel, resistant cells exhibited enhanced migratory and invasive capacities compared with parental cells, indicating acquisition of a more aggressive cellular phenotype (Fig. 1 D). To determine whether these phenotypic changes reflected altered apoptotic regulation, we profiled BCL-2 family members at the transcript and protein levels. Transcriptomic analysis revealed consistent upregulation of MCL1, BAK1, and BID and downregulation of BAX and BMF across all resistant models (Fig. 1 E, Supplementary Fig. S1 D). These changes were corroborated at the protein level: MCL-1 was increased, while NOXA and PUMA were reduced, indicating a shift toward MCL-1-dominant survival signaling with diminished apoptotic priming (Fig. 1 F, G). Dynamic BH3 profiling confirmed these altered dependencies functionally. Resistant cells showed increased mitochondrial depolarization to NOXA and BID peptides but diminished responses to the BAD peptide, directly demonstrating reduced BCL-2 dependence and compensatory MCL-1 reliance (Fig. 1 H, I). MOLM-13/R and MV4-11/R cells additionally displayed increased HRK sensitivity, implicating BCL-XL engagement, whereas OCI-AML-2/R cells exhibited a predominant MCL-1-dependent shift. Together, these data establish that acquired VEN resistance involves coordinated phenotypic diversification and context-specific rewiring of anti-apoptotic dependencies — from BCL-2 reliance toward MCL-1- and, in part, BCL-XL-mediated survival. Integrative transcriptomic and metabolomic analysis identifies ADAM15 as a main regulator of venetoclax resistance RNA sequencing of paired VEN-resistant and parental AML cell lines identified a conserved transcriptional signature comprising 27 upregulated and 222 downregulated genes shared across all three resistant models (Fig. 2 A, E). To assess clinical relevance, we interrogated the BeatAML functional genomics dataset. Differential expression analysis of ex vivo samples stratified by VEN sensitivity (AUC high vs. low) revealed extensive transcriptional reprogramming in VEN-refractory cases (Fig. 2 B). Analysis of intrinsically resistant OCI-AML-3 cells relative to VEN-sensitive lines identified an additional set of differentially expressed genes associated with primary resistance (Fig. 2 C). Cross-comparison of these datasets defined a restricted subset of concordantly regulated genes shared across experimental and clinical contexts (Fig. 2 E, F; Supplementary Tables S1, S2). KEGG pathway enrichment analysis consistently identified cell adhesion–related programs, with the cell adhesion molecule (CAM) pathway emerging as a recurrent feature across both primary and acquired resistance (Fig. 2 D, Supplementary Fig. S2 A). Given emerging evidence linking metabolic adaptation to VEN resistance, we performed untargeted metabolomic profiling of resistant and parental cells, which revealed coordinated alterations in metabolite abundance across all three models (Supplementary Fig. S2 B, C). Correlation analysis between differentially expressed genes and resistance-associated metabolites identified ADAM15 as a highly connected node, showing strong correlations with a substantial fraction of altered metabolites across all cell line pairs (|Pearson's r| > 0.8; Fig. 2 G). ADAM15 was consistently upregulated across all experimental and clinical resistance datasets, positioning it as a convergent molecular node at the interface of transcriptional and metabolic reprogramming in VEN-refractory AML. ADAM15 expression correlates with venetoclax resistance and inferior clinical outcome in AML ADAM15 expression correlates with venetoclax resistance and inferior clinical outcome in AML Having identified ADAM15 as a convergently upregulated candidate across multiple VEN-refractory contexts, we next sought to evaluate its clinical relevance in AML. To this end, we integrated ex vivo VEN sensitivity data, matched transcriptomic profiles, and clinical annotation from the BeatAML cohort (Fig. 3 A). ADAM15 expression correlated positively with VEN AUC values (Fig. 3 B) and was significantly elevated in VEN-resistant compared with VEN-sensitive samples (Fig. 3 C). Kaplan–Meier analysis of 227 AML patients receiving VEN-based therapy demonstrated that high ADAM15 expression was associated with significantly inferior overall survival (Fig. 3 D). These data indicate that elevated ADAM15 not only associates with reduced drug sensitivity but also identifies a subgroup of patients with adverse clinical outcomes in the context of VEN-based treatment. To validate these findings independently, we analyzed an institutional cohort of AML patients stratified by treatment response. ADAM15 mRNA expression was significantly higher in refractory/relapsed cases compared with those achieving remission (Fig. 3 E), a pattern corroborated at the protein level by immunoblotting (Fig. 3 F, G). Together, these data establish ADAM15 upregulation as a consistent correlate of VEN refractoriness and adverse clinical outcome across independent datasets and patient cohorts. Modulation of ADAM15 alters venetoclax sensitivity and mitochondrial apoptotic threshold in AML ADAM15 was consistently elevated in VEN-resistant AML cells at both the mRNA and protein levels (Fig. 4 A, B). To assess its functional contribution to resistance, we performed reciprocal gain- and loss-of-function studies. Ectopic ADAM15 overexpression in VEN-sensitive parental cells modestly but significantly reduced VEN sensitivity across all three lines (Fig. 4 C, D). Conversely, stable ADAM15 depletion via two independent shRNAs in the corresponding resistant derivatives partially restored VEN sensitivity (Fig. 4 E–G). These bidirectional findings identify ADAM15 as a functionally relevant modulator of VEN sensitivity in AML. In addition to drug sensitivity, ADAM15 knockdown also significantly attenuated the migratory and invasive capacities of resistant cells (Fig. 4 H, I) and reversed the aberrant cell-cycle response to VEN: whereas control resistant cells exhibited S-phase accumulation upon VEN treatment, ADAM15-depleted cells showed reduced S-phase fraction and increased sub-G₀ apoptotic population (Fig. 4 J). To investigate the molecular basis of restored sensitivity, we performed RNA sequencing in ADAM15-knockdown and control resistant cells. Although 479 shared differentially expressed genes were identified across the three models (Fig. 4 K), BCL-2 family members did not show a consistent transcriptional pattern (Fig. 4 L), suggesting that ADAM15-mediated resistance operates largely independently of uniform transcriptional reprogramming of core apoptotic machinery. We therefore assessed whether ADAM15 depletion alters mitochondrial apoptotic readiness at the functional level. Dynamic BH3 profiling revealed markedly increased mitochondrial priming in ADAM15-knockdown cells across all three resistant models, as reflected by enhanced BIM-induced depolarization (Fig. 4 M). In OCI-AML-2/R and MV4-11/R cells, increased priming extended to multiple BH3 peptides and BH3 mimetics, indicating a broad shift toward an apoptosis-permissive state. MOLM-13/R cells showed a similar pattern, with the exception of modestly reduced HRK-induced priming. These data demonstrate that ADAM15 depletion restores VEN sensitivity primarily by increasing mitochondrial apoptotic priming rather than by transcriptional reprogramming of apoptotic regulators. ADAM15 depletion enhances in vivo venetoclax efficacy and prolongs survival in resistant AML models To assess whether ADAM15 contributes to VEN resistance in vivo, we established xenograft models using parental OCI-AML-2, resistant OCI-AML-2/R scramble control, and ADAM15-knockdown OCI-AML-2/R cells (Fig. 5 A, Supplementary Fig. S3 A). VEN markedly suppressed leukemia progression in parental xenografts but was substantially attenuated in resistant scramble xenografts. ADAM15 depletion restored in vivo VEN responsiveness: mice bearing ADAM15-knockdown resistant cells showed significantly lower peripheral blood hCD45⁺ leukemic chimerism, reduced leukemic infiltration in bone marrow, liver, and spleen, and decreased spleen weight and tumor burden compared with VEN-treated resistant scramble controls (Fig. 5 B, C). Histopathological analysis confirmed reduced systemic leukemic infiltration in the ADAM15-knockdown plus VEN group (Fig. 5 D, Supplementary Fig. S3 B). Longitudinal monitoring of peripheral hCD45⁺ cells revealed that ADAM15 depletion significantly slowed leukemic expansion under VEN treatment, consistent with delayed disease progression (Fig. 5 E). Notably, ADAM15 depletion alone exerted only a limited effect on leukemic burden, whereas its combination with VEN conferred substantially greater benefit, indicating that ADAM15 knockdown primarily sensitizes resistant cells to VEN rather than broadly suppressing leukemia growth. Mice engrafted with ADAM15-knockdown resistant cells and treated with VEN exhibited significantly prolonged survival compared with VEN-treated scramble controls (Fig. 5 F). Together, these data demonstrate that ADAM15 depletion restores VEN responsiveness in vivo, suppresses leukemia progression across multiple disease compartments, and prolongs survival in resistant AML xenografts. ADAM15 drives adaptive survival signaling through HCK-dependent activation of AKT-FOXO3A axis under venetoclax stress To identify effectors downstream of ADAM15, we performed protein–protein interaction network and hub gene analyses using shared differentially expressed genes across the three ADAM15-knockdown resistant models (Supplementary Table S3 ). HCK was consistently downregulated following ADAM15 depletion and predicted to interact with ADAM15, nominating it as a candidate proximal effector (Fig. 6 A). Immunofluorescence analysis revealed increased colocalization of ADAM15 and HCK in VEN-resistant cells following VEN exposure, whereas parental cells displayed substantially weaker overlap (Fig. 6 B, Supplementary Fig. S4 A, B). Quantitative colocalization metrics confirmed enhanced spatial coupling between ADAM15 and HCK specifically in resistant cells under therapeutic stress (Fig. 6 C, D). Reciprocal co-immunoprecipitation demonstrated that ADAM15 and HCK formed a detectable complex in resistant OCI-AML-2/R cells following VEN treatment, but not in parental cells or under DMSO conditions (Fig. 6 E–G). These data indicate that ADAM15–HCK engagement is preferentially induced in resistant cells in response to VEN exposure rather than constitutively maintained. We next examined downstream signaling consequences of this interaction. Under VEN treatment, resistant cells displayed increased ADAM15 abundance together with enhanced phosphorylation of HCK, PDK1, AKT, GSK3β, and FOXO3A compared with parental cells (Fig. 6 H, I), consistent with activation of an HCK-PDK1-AKT-FOXO3A survival pathway downstream of ADAM15. VEN-treated resistant cells also showed increased p-BAD and reduced BIM expression alongside sustained MCL-1 elevation, indicating coupled anti-apoptotic remodeling (Fig. 6 H, J, Supplementary Fig. S4 E). Under basal DMSO conditions, these signaling changes were less consistent and key phosphorylation events were not robustly detected (Supplementary Fig. S4 D), indicating that VEN exposure elicits a more coherent ADAM15-associated survival response than baseline rewiring alone. Nuclear–cytoplasmic fractionation confirmed enhanced cytoplasmic retention of FOXO3A in resistant cells with a corresponding reduction in the nuclear fraction (Fig. 6 K, L), consistent with AKT-dependent suppression of FOXO3A pro-apoptotic transcriptional activity. These results define a VEN stress-induced signaling mechanism in which ADAM15 engages HCK to activate the PDK1–AKT–FOXO3A axis, driving adaptive survival signaling and anti-apoptotic remodeling in resistant AML under therapeutic stress. Pharmacologic and genetic disruption of the ADAM15-HCK axis re-sensitizes AML to venetoclax GSEA revealed global suppression of FOXO-associated transcriptional programs in resistant cells (Fig. 7 A), consistent with enhanced FOXO3A cytoplasmic sequestration in the resistant state. To determine whether this pro-survival signaling is functionally dependent on the ADAM15–HCK axis, we examined the effects of genetic ADAM15 depletion and pharmacologic HCK inhibition with PP2 in VEN-resistant OCI-AML-2/R cells. VEN treatment induced robust activation of the HCK–PDK1–AKT cascade in resistant cells, as evidenced by increased phosphorylation of HCK, PDK1, AKT, GSK3β, and FOXO3A. Both ADAM15 knockdown and PP2 treatment markedly attenuated this signaling cascade under VEN exposure (Fig. 7 B, Supplementary Fig. S4 F), indicating that sustained AKT–FOXO3A activation requires ADAM15–HCK engagement. Disruption of this axis also reversed key anti-apoptotic features of the resistant state, reducing BAD phosphorylation and restoring BIM expression (Fig. 7 B), consistent with reactivation of FOXO3A-dependent pro-apoptotic transcription. Functionally, whereas resistant cells exhibited limited apoptotic response to VEN monotherapy, both ADAM15 depletion and HCK inhibition significantly enhanced VEN-induced apoptosis, with combined treatment producing a marked increase in apoptotic cell death across multiple resistant models (Fig. 7 C). These findings establish that the ADAM15–HCK–AKT–FOXO3A axis is required for maintenance of the VEN-resistant state and is therapeutically actionable, with its disruption restoring apoptotic competence and re-sensitizing resistant AML to VEN. Discussion This study reveals that venetoclax resistance in AML is not solely a consequence of static anti-apoptotic rewiring but also depends on a dynamic, stress-responsive signaling program orchestrated by ADAM15. Under therapeutic pressure, ADAM15 engages HCK to activate AKT–FOXO3A signaling, suppressing mitochondrial apoptotic priming and sustaining the resistant state. This mechanism is therapeutically actionable: both genetic ADAM15 depletion and pharmacologic HCK inhibition restore VEN sensitivity in vitro and prolong survival in resistant xenograft models, defining the ADAM15–HCK axis as a candidate therapeutic target for VEN-refractory AML. Resistance to VEN has been attributed to multiple non-mutually exclusive mechanisms, including compensatory reliance on alternative anti-apoptotic proteins such as MCL1 or BCL-XL 7,17,18 , metabolic adaptation 19 , 20 , transcriptional and epigenetic reprogramming 21 , and activation of pro-survival kinase pathways 7 , 22 . Our findings extend current models of VEN resistance by identifying ADAM15 as a membrane-proximal signaling regulator that links therapeutic stress to adaptive kinase signaling and apoptotic remodeling. Although ADAM15 has been implicated in tumor cell adhesion, angiogenesis, and metastasis in various solid malignancies 10 – 12 , 23 , its role in hematologic drug resistance has remained unexplored. In this context, our data establish that ADAM15 contributes not only to maintenance of the resistant state but also to associated phenotypic features, including altered cell-cycle dynamics, enhanced migratory and invasive behavior, and reduced mitochondrial apoptotic priming, broadening the biological scope of ADAM15 beyond solid tumor biology. Mechanistically, activation of the ADAM15–HCK axis imposes a dual restraint on mitochondrial apoptotic competence through downstream AKT signaling. AKT-mediated BAD phosphorylation inactivates this pro-apoptotic BH3-only protein 24 , while concurrent FOXO3A phosphorylation and cytoplasmic sequestration suppresses BIM transcription — consistent with studies in hematologic malignancies linking FOXO3A inactivation to reduced pro-apoptotic transcriptional output 25 – 27 . Importantly, ADAM15–HCK engagement is not constitutive but preferentially induced under VEN exposure, distinguishing this mechanism from static anti-apoptotic rewiring and positioning it as a therapeutic stress-adaptive response. These findings carry direct translational implications. High ADAM15 expression correlated with reduced ex vivo VEN sensitivity and inferior overall survival in public datasets, observations corroborated in our institutional cohort where ADAM15 was elevated in refractory/relapsed cases — supporting its potential as a candidate biomarker of VEN response. Therapeutically, the pathway appears pharmacologically tractable. Although ADAM15 currently lacks a well-defined selective small-molecule inhibitor 28 , its oncogenic functions appear to depend mainly on scaffold-mediated signaling rather than on its metalloproteinase activity 10 – 12 , 23 . This scaffold-dependent mode of action suggests therapeutic strategies targeting protein degradation (e.g., PROTACs) or disruption of the ADAM15–HCK interface. More immediately, co-treatment with the SFK inhibitor PP2 and VEN markedly increased apoptosis in resistant cells, recapitulating genetic ADAM15 depletion. Given that SFK inhibitors (SFKi) such as dasatinib are already clinically deployed in hematologic malignancies, and that emerging evidence supports SFKi-containing regimens enhancing BCL-2-directed therapy 29 – 31 , our findings provide a mechanistic rationale for SFK-based combination strategies in VEN-refractory AML. Several limitations warrant acknowledgment. First, the downstream transcriptional programs through which FOXO3A executes this adaptive survival state remain incompletely defined and will require integrative chromatin profiling. Second, the functional relationship between this signaling axis and the metabolic reprogramming observed in resistant cells — whether downstream effector or parallel adaptation — also remains to be dissected. Finally, validation in patient-derived xenograft models will be necessary to assess translational feasibility more rigorously. In conclusion, this study identifies ADAM15 as a convergent driver of VEN resistance in AML, operating through stress-induced HCK–PDK1–AKT–FOXO3A signaling to suppress mitochondrial apoptotic priming. These findings expand the molecular framework of BCL-2 inhibitor refractoriness and provide a rationale for therapeutic strategies targeting the ADAM15–HCK axis to restore VEN sensitivity. Materials and methods Detailed information on all shRNA sequences, primer sequences, BH3 peptides, and antibodies is provided in Supplementary Table S1 . Detailed experimental protocols are provided in Supplementary Methods. Patient samples Peripheral blood or bone marrow mononuclear cells were isolated by Ficoll density gradient centrifugation from AML patients treated at Guangdong General Hospital. Samples were classified into a VEN-remission group (patients achieving CR/CRi following venetoclax-based therapy) and a VEN-refractory/relapsed group (patients failing to achieve CR/CRi or relapsing after initial response). Clinical characteristics are summarized in Supplementary Table S2 . Written informed consent was obtained in accordance with the Declaration of Helsinki. The study was approved by the Ethics Committee of Guangdong General Hospital (approval no. KY-Z-2020-551-02). Cell culture and establishment of venetoclax-resistant cells Human AML cell lines (MOLM-13, OCI-AML-2, OCI-AML-3, MV4-11, KG1α, THP-1) and HEK293T cells were maintained under standard conditions (Supplementary Methods). VEN-resistant subclones of OCI-AML-2, MV4-11, and MOLM-13 were generated through continuous exposure to stepwise increasing concentrations of ABT-199, beginning at the IC₅₀ and escalating 1.5–2-fold per cycle until cells proliferated stably at concentrations exceeding 10-fold the initial IC₅₀. Resistant cells were thereafter maintained in ABT-199-containing medium. All cell lines were authenticated by STR profiling before and after resistance induction and routinely tested for Mycoplasma contamination. Cell viability assays Cell viability was assessed by CCK-8 assay following 48-h treatment with ABT-199 or vehicle control. IC₅₀ values were determined by four-parameter nonlinear regression. All experiments were performed with at least three biological replicates in technical triplicate. Migration and invasion assays Migration and invasion were assessed using Transwell inserts with or without Matrigel coating. Cells were seeded in serum-free medium in the upper chamber, allowed to migrate toward serum-containing medium for 24 h, and quantified by flow cytometry. Flow cytometry Apoptosis was assessed by Annexin V/PI staining following ABT-199 treatment. Cell cycle distribution was analyzed by PI/RNase staining and modeled using ModFit LT. Human cell engraftment in xenograft experiments was quantified as the percentage of hCD45⁺mCD45⁻ cells among total viable CD45⁺ leukocytes. All flow cytometric analyses were performed on a Cytek Aurora Evo flow cytometer and analyzed using FlowJo (v10.8.1). Detailed staining protocols, gating strategies, and quality control criteria are provided in Supplementary Methods. Lentiviral-mediated gene knockdown and overexpression ADAM15 overexpression and shRNA knockdown constructs were delivered by lentiviral transduction into parental and resistant AML cell lines, respectively. Transduced cells were selected with puromycin, and knockdown/overexpression efficiency was verified by RT-qPCR and immunoblotting. shRNA sequences are provided in Supplementary Table S1 ; vector and transduction details are provided in Supplementary Methods. BH3 profiling Dynamic BH3 profiling was performed on digitonin-permeabilized cells using a panel of BH3 peptides (BIM, BID, HRK, NOXA, BAD, PUMA) and BH3 mimetics (ABT-199, S63845, WHI-539). Mitochondrial depolarization was monitored kinetically by JC-1 fluorescence over 2 h, and priming was calculated relative to CCCP-induced complete depolarization. Peptide sequences, concentrations, and detailed protocols are provided in Supplementary Methods. RNA extraction and real-time q-PCR Total RNA was extracted using TRIzol, reverse-transcribed, and analyzed by SYBR Green-based qPCR on an ABI 7500 system. Relative expression was normalized to GAPDH using the 2⁻ ΔΔCt method. Each biological replicate was measured in technical triplicate where indicated. RNA sequencing Poly(A)-selected RNA libraries were constructed and sequenced on an Illumina NovaSeq 6000 platform in paired-end 150 bp mode. Reads were aligned to the reference genome using HISAT2, quantified using StringTie, and differential expression analysis was performed using DESeq2 (|log₂FC| ≥ 1, adjusted P < 0.05). Raw sequencing data have been deposited in the NCBI Gene Expression Omnibus (GEO) under accession number GSEXXXXXX. (Raw sequencing data have been submitted to the GEO and are currently under processing; the accession number will be inserted prior to publication.). Untargeted metabolomics Parental and VEN-resistant cell line pairs (MOLM-13, OCI-AML-2, MV4-11) prepared in parallel with RNA-seq samples were subjected to untargeted metabolomic profiling. Metabolites were identified by accurate mass, retention time, and MS/MS fragmentation against reference databases, with differentially abundant metabolites defined by VIP > 1.0. Co-immunoprecipitation To assess the ADAM15–HCK interaction under therapeutic stress, cells were treated with whether ABT-199 or DMSO for 12 h and subjected to reciprocal co-immunoprecipitation using crosslink-based IP with antibodies against ADAM15 and HCK, with corresponding isotype controls. Bound proteins were analyzed by immunoblotting. Immunoblotting Whole-cell lysates or subcellular fractions were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with the indicated primary and HRP-conjugated secondary antibodies. For HCK inhibition studies, cells were pretreated with PP2 prior to ABT-199 exposure. Band intensities were quantified using ImageJ. Antibody details are provided in Supplementary Table S1 ; full protocols including treatment conditions, lysis buffers, and fractionation procedures are provided in Supplementary Methods. Immunofluorescence staining and confocal microscopy Cells were treated with ABT-199 or vehicle for 2 h, fixed, permeabilized with digitonin, and stained with primary antibodies followed by Alexa Fluor-conjugated secondary antibodies. Nuclei were counterstained with DAPI. Images were acquired by confocal microscopy, and colocalization was quantified using ImageJ. In vivo xenograft assays Female NOD/SCID mice (6 weeks) were intravenously injected with 4 × 10⁶ cells from one of the following groups: parental OCI-AML-2, OCI-AML-2/R, OCI-AML-2/R-shADAM15, OCI-AML-2/R-scramble, or PBS alone (cell-free control). Engraftment was confirmed at day 7 by peripheral blood hCD45 flow cytometry and Wright-Giemsa morphology, with successful engraftment defined as ≥ 1% hCD45⁺ cells. Engrafted mice were randomized (n ≥ 6 per group) to receive venetoclax (50 mg/kg, oral gavage, QD) or vehicle beginning on day 7. Disease progression was monitored weekly by peripheral blood hCD45/mCD45 chimerism. Mice were euthanized upon reaching predefined humane endpoints, and tissues were collected for flow cytometry, histology, immunohistochemistry, and molecular analyses. All procedures were approved by the IACUC (approval no. G2025074) and conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. BeatAML Cohort Analysis Clinical, transcriptomic, and drug sensitivity data were obtained from the BeatAML database 32 . Patients were stratified into venetoclax-sensitive (Q1) and venetoclax-resistant (Q4) groups by venetoclax AUC quartiles. ADAM15 expression was compared between groups and correlated with venetoclax sensitivity. Patients were further dichotomized into ADAM15-high and ADAM15-low groups using the optimal cutoff, and overall survival was analyzed by Kaplan-Meier method with log-rank testing. Statistical analysis Experiments were performed with at least three independent biological replicates, and data are presented as mean ± SD unless otherwise stated. Comparisons between two groups were made using unpaired two-tailed Student's t-test; multiple-group comparisons used one-way ANOVA with Tukey's post hoc test. IC₅₀ values were determined by four-parameter nonlinear regression. Survival was analyzed by Kaplan-Meier method with log-rank test. P < 0.05 was considered statistically significant. All analyses were performed using GraphPad Prism (v9.5) or R (v4.3.1). Declarations Data availability RNA sequencing data have been submitted to the NCBI Gene Expression Omnibus (GEO) and are currently under processing; the accession number will be inserted prior to publication. All other data supporting the findings of this study are available within the article and its supplementary files, or from the corresponding author upon reasonable request. Funding This work was supported by the Guangdong Basic and Applied Basic Research Foundation (2024B1515020054), the National Natural Science Foundation of China (82270161, 82500188), the Science and Technology Planning Project of Guangdong Province (2023B1111050004), the Project of Administration of Traditional Chinese Medicine of Guangdong Province (20242002), and the Medical Scientific Research Foundation of Guangdong Province (B2025364). Author contributions KFL and PLL conceived and designed the study. KFL, LJZ and MZX performed the experiments and analyzed the data. KFL wrote the manuscript. MZX, SXG, YLW, MW, PLL and RHX reviewed and edited the manuscript. XH, MML, PW, XMC and YTL provided the clinical samples/datasets. XD, JYW and PLL supervised and acquired funding for this study. All authors have read and approved the final manuscript. Ethics declarations This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Guangdong General Hospital (approval no. KY-Z-2020-551-02). All procedures involving animal subjects were approved by the Institutional Animal Care and Use Committee (IACUC; approval no. G2025074). Conflit of interests The authors declare no competing interests. References Short NJ, Daver N, Dinardo CD, Kadia T, Nasr LF, Macaron W et al. Azacitidine, venetoclax, and gilteritinib in newly diagnosed and relapsed or refractory FLT3-mutated AML. J Clin Oncol Off J Am Soc Clin Oncol 2024; 42: 1499–1508. Wei AH, Panayiotidis P, Montesinos P, Laribi K, Ivanov V, Kim I et al. Long-term follow-up of VIALE-C in patients with untreated AML ineligible for intensive chemotherapy. Blood 2022; 140: 2754–2756. Wei AH, Loo S, Daver N. How I treat patients with AML using azacitidine and venetoclax. Blood 2025; 145: 1237–1250. Konopleva M, Thirman MJ, Pratz KW, Garcia JS, Recher C, Pullarkat V et al. Impact of FLT3 mutation on outcomes after venetoclax and azacitidine for patients with treatment-naïve acute myeloid leukemia. Clin Cancer Res Off J Am Assoc Cancer Res 2022; 28: 2744–2752. Pratz KW, Jonas BA, Pullarkat V, Thirman MJ, Garcia JS, Döhner H et al. Long-term follow-up of VIALE-a: Venetoclax and azacitidine in chemotherapy-ineligible untreated acute myeloid leukemia. Am J Hematol 2024; 99: 615–624. Pei S, Pollyea DA, Gustafson A, Stevens BM, Minhajuddin M, Fu R et al. Monocytic subclones confer resistance to venetoclax-based therapy in patients with acute myeloid leukemia. Cancer Discov 2020; 10: 536–551. Zhang Q, Riley-Gillis B, Han L, Jia Y, Lodi A, Zhang H et al. Activation of RAS/MAPK pathway confers MCL-1 mediated acquired resistance to BCL-2 inhibitor venetoclax in acute myeloid leukemia. Signal Transduct Target Ther 2022; 7: 51. Guièze R, Liu VM, Rosebrock D, Jourdain AA, Hernández-Sánchez M, Martinez Zurita A et al. Mitochondrial reprogramming underlies resistance to BCL-2 inhibition in lymphoid malignancies. Cancer Cell 2019; 36: 369–384.e13. Montero J, Letai A. Why do BCL-2 inhibitors work and where should we use them in the clinic? Cell Death Differ 2018; 25: 56–64. Zhou J, Wang A, Cai T, Li Y, Du W, Zhang Y et al. Integrin α3/α6 and αV are implicated in ADAM15-activated FAK and EGFR signalling pathway individually and promote non-small-cell lung cancer progression. Cell Death Dis 2022; 13: 486. Cabañas C, Rossi E, Bartolomé RA, Doberstein K, Altevogt P, Casal JI et al. The emerging role of human transmembrane RGD-based counter-receptors of integrins in health and disease. Cell Mol Biol Lett 2025; 30: 110. Evers M, Stühmer T, Schreder M, Steinbrunn T, Rudelius M, Jundt F et al. Association of ADAM family members with proliferation signaling and disease progression in multiple myeloma. Blood Cancer J 2024; 14: 156. Ciechomska IA, Gielniewski B, Wojtas B, Kaminska B, Mieczkowski J. EGFR/FOXO3a/BIM signaling pathway determines chemosensitivity of BMP4-differentiated glioma stem cells to temozolomide. Exp Mol Med 2020; 52: 1326–1340. Liu Y, Wang Y, Li X, Jia Y, Wang J, Ao X. FOXO3a in cancer drug resistance. Cancer Lett 2022; 540: 215724. Gong K, Xu B, Gong L, Zhong M, Han C, Liu Y et al. ITGA2 mediates the resistance of hepatocellular carcinoma to lenvatinib by activating the AKT/FOXO3A signaling pathway. Cancers 2025; 17. doi: 10.3390/cancers17172846 . Ma J, Zhao S, Qiao X, Knight T, Edwards H, Polin L et al. Inhibition of bcl-2 synergistically enhances the antileukemic activity of midostaurin and gilteritinib in preclinical models of FLT3-mutated acute myeloid leukemia. Clin Cancer Res Off J Am Assoc Cancer Res 2019; 25: 6815–6826. Kuusanmäki H, Dufva O, Vähä-Koskela M, Leppä A-M, Huuhtanen J, Vänttinen I et al. Erythroid/megakaryocytic differentiation confers BCL-XL dependency and venetoclax resistance in acute myeloid leukemia. Blood 2023; 141: 1610–1625. Pan R, Hogdal LJ, Benito JM, Bucci D, Han L, Borthakur G et al. Selective BCL-2 inhibition by ABT-199 causes on-target cell death in acute myeloid leukemia. Cancer Discov 2014; 4: 362–375. Chen X, Glytsou C, Zhou H, Narang S, Reyna DE, Lopez A et al. Targeting mitochondrial structure sensitizes acute myeloid leukemia to venetoclax treatment. Cancer Discov 2019; 9: 890–909. Roca-Portoles A, Rodriguez-Blanco G, Sumpton D, Cloix C, Mullin M, Mackay GM et al. Venetoclax causes metabolic reprogramming independent of BCL-2 inhibition. Cell Death Dis 2020; 11: 616. Thomalla D, Beckmann L, Grimm C, Oliverio M, Meder L, Herling CD et al. Deregulation and epigenetic modification of BCL2-family genes cause resistance to venetoclax in hematologic malignancies. Blood 2022; 140: 2113–2126. Satta T, Li L, Chalasani SL, Hu X, Nkwocha J, Sharma K et al. Dual mTORC1/2 inhibition synergistically enhances AML cell death in combination with the BCL2 antagonist venetoclax. Clin Cancer Res Off J Am Assoc Cancer Res 2023; 29: 1332–1343. Duhachek-Muggy S, Qi Y, Wise R, Alyahya L, Li H, Hodge J et al. Metalloprotease-disintegrin ADAM12 actively promotes the stem cell-like phenotype in claudin-low breast cancer. Mol Cancer 2017; 16: 32. Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y et al. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 1997; 91: 231–241. Gilley J, Coffer PJ, Ham J. FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons. J Cell Biol 2003; 162: 613–622. Dijkers PF, Medema RH, Lammers JW, Koenderman L, Coffer PJ. Expression of the pro-apoptotic bcl-2 family member bim is regulated by the forkhead transcription factor FKHR-L1. Curr Biol CB 2000; 10: 1201–1204. Rosich L, Saborit-Villarroya I, López-Guerra M, Xargay-Torrent S, Montraveta A, Aymerich M et al. The phosphatidylinositol-3-kinase inhibitor NVP-BKM120 overcomes resistance signals derived from microenvironment by regulating the akt/FoxO3a/bim axis in chronic lymphocytic leukemia cells. Haematologica 2013; 98: 1739–1747. Chung Y-L, Pan C-H, Wang CC-N, Hsu K-C, Sheu M-J, Chen H-F et al. Methyl protodioscin, a steroidal saponin, inhibits neointima formation in vitro and in vivo. J Nat Prod 2016; 79: 1635–1644. Moujalled DM, Hanna DT, Hediyeh-Zadeh S, Pomilio G, Brown L, Litalien V et al. Cotargeting BCL-2 and MCL-1 in high-risk B-ALL. Blood Adv 2020; 4: 2762–2767. Garciaz S, Montersino C, Bourgoin M, Jacquel A, Castellano R, Guille A et al. Dasatinib overcomes AML cells resistant to BCL2 inhibition by degrading MCL1. Br J Haematol 2025; 207: 381–386. Gocho Y, Liu J, Hu J, Yang W, Dharia NV, Zhang J et al. Network-based systems pharmacology reveals heterogeneity in LCK and BCL2 signaling and therapeutic sensitivity of T-cell acute lymphoblastic leukemia. Nat Cancer 2021; 2: 284–299. Tyner JW, Tognon CE, Bottomly D, Wilmot B, Kurtz SE, Savage SL et al. Functional genomic landscape of acute myeloid leukaemia. Nature 2018; 562: 526–531. Additional Declarations There is NO conflict of interest to disclose. Supplementary Files SupFigure.pdf Supplemental Figure SupMethods.pdf Supplemental Methods TableS1.xlsx Supplemental Table 1 TableS2.xlsx Supplemental Table 2 TableS3.xlsx Supplemental Table 3 Cite Share Download PDF Status: Under Review Version 1 posted Reviewer # 1 agreed at journal 08 May, 2026 Reviewers invited by journal 07 May, 2026 Editor assigned by journal 23 Apr, 2026 Submission checks completed at journal 23 Apr, 2026 First submitted to journal 22 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9498017","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":636461342,"identity":"448d29db-72a8-4dec-b41d-6bdf754ca7b9","order_by":0,"name":"Peilong Lai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIie3RPQrCMBiA4ZRAukSzplh6ho4ep1nsUqGTZCiSQdpB3Dt4CEd3IVNcpWN7BDdBENOfuckomHf4suQhCQHA5frBlsATeqGhHl6b8MJM0ESwHjBulbQhYz1BQXeAFsS/CZjzNSar6sGZQIBUx2SeYCZgrSgOzmrXsGsIqLpfDBfTZFFSHDfZpmEKgZhuDYR0mnxGkrMSWhDanyJ6kkpgSfQptdRvqTNIEyWx8S2EpC3Mi31EaNo9X7yISHWaJzr/Pa44HqZp+9D0fX5rtdvlcrn+ry9CykAKvlPnXQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-0256-1082","institution":"Guangdong General Hospital, Guangdong Academy of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Peilong","middleName":"","lastName":"Lai","suffix":""},{"id":636461343,"identity":"89381b0e-3b59-418e-b857-5d882e7a1126","order_by":1,"name":"Kaifan Liu","email":"","orcid":"https://orcid.org/0009-0009-3787-5226","institution":"Guangdong General Hospital, Guangdong Academy of Medical 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Du","email":"","orcid":"https://orcid.org/0000-0001-7089-0131","institution":"Guangdong Provincial People's Hospital (Guangdong Academy of Medical Sciences), Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Du","suffix":""},{"id":636461356,"identity":"a4f596cb-de21-45da-a4da-b04137c16ac0","order_by":14,"name":"Jianyu Weng","email":"","orcid":"https://orcid.org/0000-0001-5446-292X","institution":"Guangdong General Hospital/Guangdong Academy of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jianyu","middleName":"","lastName":"Weng","suffix":""}],"badges":[],"createdAt":"2026-04-22 15:00:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9498017/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9498017/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109799616,"identity":"3786cccf-4b3e-4f0b-b6e6-238a074538aa","added_by":"auto","created_at":"2026-05-22 15:32:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5902478,"visible":true,"origin":"","legend":"\u003cp\u003eVenetoclax-resistant AML cells exhibit attenuated apoptotic responses, enhanced migratory and invasive capacities, and rewired anti-apoptotic dependencies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Dose–response curves of parental and venetoclax (VEN)-resistant AML cells treated with increasing concentrations of ABT-199 for 48 h. \u003cstrong\u003e(B)\u003c/strong\u003e Apoptosis in parental and VEN-resistant AML cells following treatment with 1 μM or 10 μM ABT-199 for 24 h, assessed by Annexin V/propidium iodide (PI) staining. Total apoptosis represents the sum of early and late apoptotic cells. \u003cstrong\u003e(C)\u003c/strong\u003e Cell-cycle distribution of parental and VEN-resistant AML cells following treatment with ABT-199 or vehicle control. \u003cstrong\u003e(D)\u003c/strong\u003e Quantification of Transwell invasion (left) and migration (right) in parental and VEN-resistant AML cells. \u003cstrong\u003e(E)\u003c/strong\u003eHeatmap of RNA-seq analysis showing differential expression of apoptosis-related genes in VEN-resistant cells relative to parental controls, presented as log₂(fold change) based on TPM-normalized counts.\u003cstrong\u003e (F, G) \u003c/strong\u003eRepresentative immunoblots \u003cstrong\u003e(G)\u003c/strong\u003e and corresponding densitometric quantification (\u003cstrong\u003eF\u003c/strong\u003e) of anti- and pro-apoptotic BCL-2 family proteins. Protein levels were normalized to GAPDH and expressed relative to parental controls.\u003cstrong\u003e (H) \u003c/strong\u003eRepresentative JC-1 fluorescence images indicating mitochondrial membrane potential (ΔΨm). Green fluorescence indicates JC-1 monomers (depolarized mitochondria), whereas orange-red fluorescence indicates JC-1 aggregates (polarized mitochondria). Scale bar, 20 μm. \u003cstrong\u003e(I)\u003c/strong\u003e Heatmap of dynamic BH3 profiling showing Δ% priming in response to the indicated BH3 peptides and BH3 mimetics. Δ% priming was calculated as the difference in priming between resistant and corresponding parental cells.\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SD from three independent biological replicates. Statistical significance is indicated as follows: *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001, and ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001. For transcriptomic analyses, adjusted P values (\u003cem\u003eP\u003c/em\u003eadj) are indicated using the same thresholds.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-9498017/v1/65876bb0e3554c6944ae2e67.png"},{"id":109759637,"identity":"aa0adf69-0445-42ae-9934-a24cfb15bfea","added_by":"auto","created_at":"2026-05-22 07:27:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5938549,"visible":true,"origin":"","legend":"\u003cp\u003eIntegrative multi-omic analyses identify ADAM15 as a convergently upregulated feature across venetoclax-resistant AML states.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A–C) \u003c/strong\u003eVolcano plots depicting differentially expressed genes (DEGs) across distinct models of venetoclax (VEN) resistance: \u003cstrong\u003e(A)\u003c/strong\u003e paired VEN-resistant versus parental AML cell lines; \u003cstrong\u003e(B) \u003c/strong\u003eprimary AML samples stratified by VEN response in the BeatAML cohort; and \u003cstrong\u003e(C)\u003c/strong\u003e intrinsically VEN-insensitive versus VEN-sensitive AML cell lines. In (B), samples were dichotomized based on the upper and lower quartiles of VEN area under the curve (AUC), corresponding to VEN-refractory (high AUC) and VEN-sensitive (low AUC) groups. Significantly upregulated and downregulated genes are highlighted in red and blue, respectively. \u003cstrong\u003e(D)\u003c/strong\u003e KEGG pathway enrichment analysis of DEGs derived from clinical and experimental resistance models. \u003cstrong\u003e(E, F)\u003c/strong\u003e Identification of a conserved resistance-associated transcriptional program. Venn diagrams illustrate the overlap of downregulated \u003cstrong\u003e(E)\u003c/strong\u003e and upregulated \u003cstrong\u003e(F) \u003c/strong\u003eDEGs across experimental systems and patient-derived datasets. \u003cstrong\u003e(G)\u003c/strong\u003eIntegrative correlation analysis of resistance-associated DEGs and differentially abundant metabolites. ADAM15 emerges as a highly connected node, exhibiting strong correlations (|Pearson’s r| \u0026gt; 0.8) with a substantial subset of resistance-associated metabolites across all three VEN-resistant models.\u003c/p\u003e\n\u003cp\u003eRNA-seq and untargeted metabolomics were performed with n = 3 independent biological replicates per group. For transcriptomic analyses, significance was defined as adjusted P value (\u003cem\u003ePadj\u003c/em\u003e) \u0026lt; 0.05 and |log₂ fold change| \u0026gt; 1. Clinical data were obtained from the BeatAML functional genomics resource.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-9498017/v1/da47c92d3d2f314b4c7db567.png"},{"id":109759581,"identity":"931bb883-683e-423c-9de0-51d27ad6f4d5","added_by":"auto","created_at":"2026-05-22 07:27:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":772119,"visible":true,"origin":"","legend":"\u003cp\u003eADAM15 expression correlates with venetoclax resistance and inferior clinical outcomes in AML patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Schematic workflow showing the integrative analysis of the BeatAML dataset, encompassing transcriptomic profiling, \u003cem\u003eex vivo\u003c/em\u003e drug sensitivity, and clinical survival outcomes. \u003cstrong\u003e(B)\u003c/strong\u003e Pearson correlation analysis showing positive correlation between \u003cem\u003eADAM15\u003c/em\u003e mRNA expression and Venetoclax AUC values in the BeatAML cohort (R = 0.46,p=2.8×10^(-10)). \u003cstrong\u003e(C)\u003c/strong\u003e Comparison of \u003cem\u003eADAM15\u003c/em\u003e expression levels between the Venetoclax-sensitive (Bottom 25% AUC, n=42) and Venetoclax-resistant (Top 25% AUC, n=42) groups. Distribution of AUC values with quartile cutoffs is shown in the lower panel. p=1.23×10^(-7) by Mann-Whitney U test.\u003cstrong\u003e (D)\u003c/strong\u003e Kaplan-Meier survival curves showing overall survival (OS) of AML patients stratified by \u003cem\u003eADAM15\u003c/em\u003eexpression levels (High vs. Low). \u003cem\u003ep\u003c/em\u003e = 0.047 by log-rank test. \u003cstrong\u003e(E)\u003c/strong\u003eQuantitative RT-PCR analysis of \u003cem\u003eADAM15\u003c/em\u003e mRNA levels in an independent institutional cohort of AML patients who achieved remission (n=5) versus those with refractory/relapsed disease (n=6) following VEN-based therapy. Data were normalized to GAPDH. \u003cstrong\u003e(F, G)\u003c/strong\u003e Representative immunoblots\u003cstrong\u003e (F)\u003c/strong\u003e and densitometric quantification \u003cstrong\u003e(G) \u003c/strong\u003eof ADAM15 protein levels in the remission and refractory/relapsed patient groups. GAPDH served as the loading control.\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SD. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 by Student’s t-test (E, G). VEN, venetoclax; AUC, area under the curve; mRNA, messenger RNA.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-9498017/v1/14f3b099398575605af5bce2.png"},{"id":109759729,"identity":"1a1b960e-a285-4b7a-93c7-5d54559457da","added_by":"auto","created_at":"2026-05-22 07:27:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":647993,"visible":true,"origin":"","legend":"\u003cp\u003eADAM15 modulation alters venetoclax sensitivity through transcriptional reprogramming and mitochondrial priming dynamics in resistant AML cells\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Relative ADAM15 mRNA expression in parental (Par) and venetoclax-resistant (Res) MOLM-13, OCI-AML-2, and MV4-11 cells, quantified by RT-qPCR and normalized to the respective parental control (set to 1). \u003cstrong\u003e(B)\u003c/strong\u003e Representative immunoblots and densitometric quantification of ADAM15 protein abundance in parental and venetoclax-resistant derivatives across all three AML cell lines. GAPDH served as the loading control. \u003cstrong\u003e(C, D)\u003c/strong\u003e Ectopic ADAM15 overexpression in MOLM-13, OCI-AML-2, and MV4-11 cells was confirmed by immunoblotting (C). Cell viability of ADAM15-overexpressing and empty-vector control cells was assessed following 48-hour exposure to increasing concentrations of ABT-199 (D). \u003cstrong\u003e(E, F)\u003c/strong\u003eStable ADAM15 knockdown efficiency in MOLM-13/R, OCI-AML-2/R, and MV4-11/R cells transduced with two independent shRNA constructs (sh#1 and sh#2) or a non-targeting scramble control, confirmed at the mRNA level by RT-qPCR (E) and at the protein level by immunoblotting with densitometric quantification (F). \u003cstrong\u003e(G)\u003c/strong\u003eCell viability dose–response curves of shADAM15 and scramble-control MOLM-13/R, OCI-AML-2/R, and MV4-11/R cells treated with increasing concentrations of ABT-199 for 48 hours.\u003cstrong\u003e (H, I)\u003c/strong\u003e Transwell migration (H) and Matrigel invasion (I) assays in shADAM15 and scramble-control resistant AML cells. Migratory and invasive capacity is expressed as the percentage of input cells that traversed the membrane.\u003cstrong\u003e (J)\u003c/strong\u003e Cell-cycle distribution in shADAM15 and scramble-control cells following 24-hour treatment with venetoclax (10 µM) or DMSO vehicle, assessed by propidium iodide staining and flow cytometry. Sub-G₀, G₀/G₁, S, and G₂/M fractions are presented as stacked bar graphs.\u003cstrong\u003e (K)\u003c/strong\u003e Three-way Venn diagram depicting the overlap of differentially expressed genes (DEGs) identified by RNA sequencing in each ADAM15-knockdown resistant model relative to its respective scramble control. The intersection contains 479 genes concordantly dysregulated across all three models (adjusted \u003cem\u003eP\u003c/em\u003e ≤ 0.05; |log₂FC| ≥ 1.0).\u003cstrong\u003e (L)\u003c/strong\u003e Heatmap of log₂(fold change) values for selected BCL-2 family members and BH3-only proteins derived from RNA sequencing of ADAM15-knockdown versus scramble-control resistant cells across the three models. \u003cstrong\u003e(M)\u003c/strong\u003eDynamic BH3 profiling heatmap depicting the change in mitochondrial depolarization (Δ% priming) in response to a panel of BH3 peptides and venetoclax (VEN) in ADAM15-knockdown resistant cells (MOLM-13/R KD, OCI-AML-2/R KD, and MV4-11/R KD) relative to scramble controls. Mitochondrial membrane potential was measured kinetically using JC-1 dye (1 µM) at 37°C, with fluorescence acquisition every 5 minutes over a 2-hour period. Warmer colors indicate greater induction of mitochondrial priming.\u003c/p\u003e\n\u003cp\u003eAll data represent mean ± SD from at least three independent biological replicates. Two-group comparisons were performed using an unpaired two-tailed Student's \u003cem\u003et\u003c/em\u003e-test; multi-group comparisons were assessed by one-way ANOVA with Tukey's post hoc correction. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-9498017/v1/e71ea6c736c733997e2f2bd0.png"},{"id":109799477,"identity":"6cb88985-4c81-4dff-b425-a88d6a935718","added_by":"auto","created_at":"2026-05-22 15:29:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1198776,"visible":true,"origin":"","legend":"\u003cp\u003eADAM15 depletion enhances venetoclax efficacy and prolongs survival in venetoclax-resistant AML xenograft models.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Schematic of the in vivo experimental design. Female NOD/SCID mice (6 weeks old) were engrafted via tail-vein injection with 4 × 10⁶ cells or cell-free PBS on day 0. Starting on day 7, mice received ABT-199 (50 mg/kg daily, administered orally, formulated in 10% DMSO/corn oil) or vehicle (10% DMSO/corn oil). Weekly body weight measurements, biweekly submandibular blood sampling (50 µL), and continuous humane endpoint monitoring were performed throughout the study. Human CD45⁺ (hCD45⁺) chimerism was quantified by flow cytometry. All surviving animals were euthanized on day 69; tissues were then harvested, weighed, and processed for downstream analyses. \u003cstrong\u003e(B)\u003c/strong\u003e Liver weight (left) and spleen weight (right) at endpoint stratified by cell injection status, venetoclax resistance phenotype, ADAM15 knockdown, and treatment regimen. Each symbol represents an individual mouse (n ≥ 6 per group); horizontal bars indicate mean ± SD. \u003cstrong\u003e(C)\u003c/strong\u003eLongitudinal peripheral blood hCD45⁺ chimerism monitored biweekly by flow cytometry across experimental groups. \u003cstrong\u003e(D)\u003c/strong\u003e Representative hematoxylin and eosin–stained sections of liver, spleen, and leukemic infiltrates (top three rows; scale bars, 500 µm) and immunohistochemical staining for ADAM15 (15 µg/mL; bottom row; scale bars, 20 µm) in tumor sections from indicated experimental groups. N.D., not detected. \u003cstrong\u003e(E)\u003c/strong\u003e hCD45⁺ leukemic burden quantified by flow cytometry at endpoint in peripheral blood (PB), bone marrow (BM), liver, and spleen across experimental groups (n ≥ 6 per group).\u003cstrong\u003e (F)\u003c/strong\u003e Kaplan–Meier survival curves for mice receiving different treatments. Cell-free PBS-injected animals served as non-engraftment controls. Survival differences were evaluated by log-rank test (n ≥ 6 per group).\u003c/p\u003e\n\u003cp\u003eAll data are presented as mean ± SD. Two-group comparisons were performed using unpaired Student's \u003cem\u003et\u003c/em\u003e-tests; multi-group comparisons used one-way ANOVA with Tukey's \u003cem\u003epost hoc\u003c/em\u003e test. Survival analysis employed the log-rank test. Statistical significance was defined as follows: *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001; ****p \u0026lt; 0.0001; ns, not significant.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-9498017/v1/fdee11899b64a67198cc70dc.png"},{"id":109759946,"identity":"d00a55b5-140f-4949-944a-9cb83bb8a83c","added_by":"auto","created_at":"2026-05-22 07:27:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1111416,"visible":true,"origin":"","legend":"\u003cp\u003eADAM15 drives adaptive survival signaling through HCK-dependent activation of the AKT–FOXO3A axis under venetoclax stress.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Protein–protein interaction (PPI) network derived from hub gene analysis of 479 differentially expressed genes shared across three ADAM15-knockdown venetoclax-resistant models. \u003cstrong\u003e(B)\u003c/strong\u003e Representative confocal immunofluorescence images depicting colocalization of ADAM15 (AF488, green) and HCK (AF647, red) in MOLM-13/R, OCI-AML-2/R, and MV4-11/R cells following 2-hour exposure to venetoclax (10 µM). Insets show magnified views of the boxed regions. Fluorescence intensity profiles along the indicated scan lines (right panels) illustrate the spatial overlap between ADAM15 and HCK signals. \u003cstrong\u003e(C)\u003c/strong\u003e Quantification of Manders' thresholded overlap coefficients (tM2), representing the fraction of ADAM15 signal overlapping with HCK, in venetoclax- versus DMSO-treated resistant (Res) and parental (Par) cells across MOLM-13 (left), OCI-AML-2 (middle), and MV4-11 (right) models. Each data point represents a single cell analyzed across at least three independent experiments (minimum five fields per experiment). \u003cstrong\u003e(D)\u003c/strong\u003ePixel-by-pixel fluorescence intensity scatter plots depicting correlation between HCK-AF647 and ADAM15-AF488 signals in resistant and parental MOLM-13, OCI-AML-2, and MV4-11 cells treated with venetoclax (10 µM) or DMSO for 2 hours. Pearson's correlation coefficients (R) are indicated within each panel. \u003cstrong\u003e(E–G)\u003c/strong\u003eReciprocal co-immunoprecipitation (co-IP) analysis in OCI-AML-2/R cells treated for 2 hours with venetoclax (10 µM; \u003cstrong\u003eE\u003c/strong\u003e) or DMSO (\u003cstrong\u003eF\u003c/strong\u003e), and in parental OCI-AML-2 cells treated with venetoclax (10 µM; \u003cstrong\u003eG\u003c/strong\u003e). Immunoprecipitations were performed using anti-ADAM15, anti-HCK, goat IgG (Gt), or rabbit IgG (Rb) isotype control antibodies, as indicated. Precipitated complexes were probed by immunoblot for ADAM15 (93 kDa) and HCK (61 kDa). Whole-cell lysate (Input) is shown as reference. \u003cstrong\u003e(H)\u003c/strong\u003e Immunoblot analysis of AKT pathway components and BCL-2 family members in parental (Par) and venetoclax-resistant (Res) MOLM-13, OCI-AML-2, and MV4-11 cells following 12-hour venetoclax treatment. GAPDH served as the loading control. \u003cstrong\u003e(I)\u003c/strong\u003eDensitometric quantification of p-AKT (Thr308), p-GSK-3β, and p-FOXO3A relative to total FOXO3A from immunoblots in panel H, expressed as fold change normalized to the respective parental control. \u003cstrong\u003e(J)\u003c/strong\u003e Densitometric quantification of BIM, p-BAD, and BID protein levels from immunoblots in panel H, expressed as fold change normalized to the respective parental control. \u003cstrong\u003e(K)\u003c/strong\u003eRelative nuclear and cytoplasmic distribution of FOXO3A in parental and resistant OCI-AML-2 cells, expressed as the fraction of total FOXO3A signal within each compartment. \u003cstrong\u003e(L)\u003c/strong\u003e Representative immunoblots of FOXO3A in whole-cell lysate (WCL), nuclear (Nuc), and cytoplasmic (Cyt) fractions from parental and OCI-AML-2/R cells. Lamin B1 and GAPDH served as nuclear and cytoplasmic compartment controls, respectively.\u003c/p\u003e\n\u003cp\u003eAll quantitative data are presented as mean ± SD from at least three independent biological replicates. Two-group comparisons were performed using an unpaired two-tailed Student's \u003cem\u003et\u003c/em\u003e-test. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001; ns, not significant.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-9498017/v1/0e115239c52f37093102d83a.png"},{"id":109439872,"identity":"9ad50d7d-273d-4e4f-b990-f842c8ae15cc","added_by":"auto","created_at":"2026-05-18 07:02:54","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1337263,"visible":true,"origin":"","legend":"\u003cp\u003eADAM15 depletion abrogates HCK phosphorylation and AKT–FOXO3A activation, restoring venetoclax sensitivity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eGSEA enrichment plot for the FOXO signaling pathway (KEGG: hsa04068) derived from transcriptomic profiling of three matched parental/resistant cell line pairs following 12 h venetoclax treatment. The enrichment score profile (green curve), leading-edge gene positions (black vertical bars), and ranked list metric (grey bars) are shown. Genes positively and negatively correlated with the resistant signature are indicated in red and blue, respectively. The zero-crossing point is annotated. \u003cstrong\u003e(B)\u003c/strong\u003e Immunoblot analysis of HCK–AKT–FOXO3A pathway components and BCL-2 family members in OCI-AML-2/R cells under the following conditions: parental versus resistant background; venetoclax versus vehicle; scramble versus ADAM15 shRNA; and PP2 (10 µM, HCK inhibitor) versus vehicle. GAPDH served as the loading control. Bracket annotations denote HCK–AKT–FOXO3A signaling components and BCL-2 family members, respectively. \u003cstrong\u003e(C)\u003c/strong\u003e Total apoptosis rates (%) in MOLM-13/R, OCI-AML-2/R, and MV4-11/R cells subjected to ADAM15 knockdown (ADAM15-KD), combined venetoclax and PP2 treatment (VEN+PP2; 10 µM), venetoclax alone (VEN), or PP2 alone (10 µM), as assessed by annexin V/propidium iodide staining and flow cytometry. \u003cstrong\u003e(D)\u003c/strong\u003e Schematic model illustrating the mechanistic basis of venetoclax sensitivity versus resistance in AML. In sensitive cells (\u003cem\u003eleft\u003c/em\u003e), venetoclax disrupts BCL-2–mediated sequestration of pro-apoptotic proteins, liberating BAX/BAK to induce mitochondrial outer membrane permeabilization (MOMP), cytochrome \u003cem\u003ec\u003c/em\u003erelease, and caspase-9/caspase-3–dependent apoptosis. In resistant cells (\u003cem\u003eright\u003c/em\u003e), upregulated ADAM15 engages HCK and drives sequential phosphorylation of PDK1, AKT, and GSK-3β. Activated AKT phosphorylates FOXO3A, promoting its cytoplasmic sequestration and consequent suppression of pro-apoptotic target gene transcription, including \u003cem\u003eBIM\u003c/em\u003e downregulation. Concurrent BAD phosphorylation further attenuates pro-apoptotic signaling. Collectively, these adaptive events sustain cell survival, cell cycle progression, and metabolic reprogramming despite continuous venetoclax exposure.\u003c/p\u003e\n\u003cp\u003eAll data are presented as mean ± SD from at least three independent biological replicates. Comparisons among multiple groups were assessed by one-way ANOVA followed by Tukey's post hoc test. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001; \u003cem\u003ens\u003c/em\u003e, not significant.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-9498017/v1/3a570f0beda4747f3d45e811.png"},{"id":109759560,"identity":"5c57c271-0cb0-410f-a3d2-8ecfcf350cdd","added_by":"auto","created_at":"2026-05-22 07:27:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1462246,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9498017/v1/a36d217d-a8fb-4e58-b0f4-e7cfd3c66e21.pdf"},{"id":109439863,"identity":"6d5979b3-c410-49d3-8385-cfb39086df47","added_by":"auto","created_at":"2026-05-18 07:02:54","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":870514,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Figure\u003c/p\u003e","description":"","filename":"SupFigure.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9498017/v1/bc7fd6039aa4ebc6ea5512be.pdf"},{"id":109439866,"identity":"942b7f26-4304-4624-93ea-ee71ef2a3733","added_by":"auto","created_at":"2026-05-18 07:02:54","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":184426,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Methods\u003c/p\u003e","description":"","filename":"SupMethods.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9498017/v1/106466d4c513e4e6f776c7cf.pdf"},{"id":109759577,"identity":"8b09b70e-4390-4056-bb34-66f90220c673","added_by":"auto","created_at":"2026-05-22 07:27:22","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":20394,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Table 1\u003c/p\u003e","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9498017/v1/3bba168fcb39d496c8a09462.xlsx"},{"id":109759455,"identity":"494e26b5-0a33-43f9-99d7-fdb1fc9063b8","added_by":"auto","created_at":"2026-05-22 07:27:06","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":17759,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Table 2\u003c/p\u003e","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9498017/v1/484ae95a9d539ef657278635.xlsx"},{"id":109759536,"identity":"4b67b6e5-dd4f-42ab-9234-adc0e1f31920","added_by":"auto","created_at":"2026-05-22 07:27:17","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":10553,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Table 3\u003c/p\u003e","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9498017/v1/27360722bd59fc3a95c69de9.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"ADAM15 drives venetoclax resistance via HCK–AKT–FOXO3A signaling in AML","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAcute myeloid leukemia (AML) remains a leading cause of leukemia-related mortality in adults. The BCL-2 inhibitor venetoclax (VEN), combined with hypomethylating agents or low-dose chemotherapy, has substantially improved initial response rates in treatment-ineligible patients; however, responses are frequently transient and heterogeneous\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, and median overall survival remains only 9\u0026ndash;14 months\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The near-universal emergence of resistance is therefore the principal barrier to durable clinical benefit.\u003c/p\u003e \u003cp\u003eAt the mechanistic level, VEN resistance converges on mitochondrial apoptotic rewiring: resistant AML cells reduce BCL-2 dependence and shift survival reliance toward MCL-1 or BCL-XL\u003csup\u003e6,7\u003c/sup\u003e, reflected functionally by diminished mitochondrial priming and impaired apoptosis induction\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. While these downstream adaptations are well characterized, the upstream signaling mechanisms that dynamically orchestrate them under therapeutic stress remain poorly defined, limiting the development of rational combination strategies.\u003c/p\u003e \u003cp\u003eA Disintegrin And Metalloproteinase 15 (ADAM15) is a multifunctional transmembrane protein that acts predominantly as a signaling scaffold to promote tumor progression and therapeutic resistance. In solid tumors, ADAM15 drives therapeutic resistance by activating EGFR- and Src family kinase-dependent pro-survival pathways and recruiting kinase-active signaling complexes under stress\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Despite these observations, its role in hematologic malignancies\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e \u0026mdash; and specifically in AML therapy resistance \u0026mdash; remains largely unexplored. Notably, AKT\u0026ndash;FOXO3A signaling, a candidate effector axis downstream of scaffold-mediated kinase activation, is activated in adverse-risk AML and associated with inferior responses to VEN-based regimens\u003csup\u003e\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, yet whether this axis is engaged in response to VEN treatment and how it interfaces with mitochondrial apoptotic rewiring remain unknown.\u003c/p\u003e \u003cp\u003eIntegrating multi-omics data across experimental models and clinical cohorts, we identify ADAM15 as a convergent driver of VEN resistance that engages HCK to activate AKT\u0026ndash;FOXO3A signaling and suppress mitochondrial apoptotic priming. Genetic and pharmacologic disruption of this axis restores VEN sensitivity in vitro and in vivo, defining the ADAM15\u0026ndash;HCK\u0026ndash;AKT\u0026ndash;FOXO3A pathway as a therapeutically actionable vulnerability for overcoming BCL-2 inhibitor resistance in AML.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eVenetoclax resistance in AML is driven by phenotypic diversification and anti-apoptotic rewiring\u003c/h2\u003e\n \u003cp\u003eSensitivity profiling across a panel of human AML cell lines identified MOLM-13, OCI-AML-2, MV4-11, and KG1\u0026alpha; as VEN-sensitive, and OCI-AML-3 and THP-1 as intrinsically resistant (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Stepwise dose escalation in three sensitive lines generated resistant derivatives (MOLM-13/R, OCI-AML-2/R, MV4-11/R) with \u0026gt;\u0026thinsp;10-fold increases in IC₅₀ (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB).\u003c/p\u003e\n \u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eIC50 values and fold changes of Venetoclax in parental and resistant AML cell lines.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eCell Line\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eParental IC50 (\u0026micro;M) [95% CI]\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eResistant IC50 (\u0026micro;M) [95% CI]\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003eFold Change\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eMOLM-13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\n \u003cp\u003e0.29 [0.26\u0026ndash;0.34]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e6.59 [5.44\u0026ndash;7.95]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e22.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eOCI-AML-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\n \u003cp\u003e0.04 [0.036\u0026ndash;0.045]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e14.02 [11.00\u0026ndash;18.15]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e345.24\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eMV4-11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\n \u003cp\u003e0.03 [0.026\u0026ndash;0.032]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e2.28 [1.72\u0026ndash;3.07]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e79.19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eVEN-resistant cells displayed markedly attenuated apoptotic responses to ABT-199 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). Cell-cycle analysis revealed a divergent response to drug exposure: parental cells underwent S-phase reduction and sub-G0 accumulation, whereas resistant cells evaded cell-cycle restraint and maintained S-phase progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). These findings suggest that VEN resistance is associated with a fundamentally altered proliferative response to drug exposure. In parallel, resistant cells exhibited enhanced migratory and invasive capacities compared with parental cells, indicating acquisition of a more aggressive cellular phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e\n \u003cp\u003eTo determine whether these phenotypic changes reflected altered apoptotic regulation, we profiled BCL-2 family members at the transcript and protein levels. Transcriptomic analysis revealed consistent upregulation of MCL1, BAK1, and BID and downregulation of BAX and BMF across all resistant models (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD). These changes were corroborated at the protein level: MCL-1 was increased, while NOXA and PUMA were reduced, indicating a shift toward MCL-1-dominant survival signaling with diminished apoptotic priming (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, G).\u003c/p\u003e\n \u003cp\u003eDynamic BH3 profiling confirmed these altered dependencies functionally. Resistant cells showed increased mitochondrial depolarization to NOXA and BID peptides but diminished responses to the BAD peptide, directly demonstrating reduced BCL-2 dependence and compensatory MCL-1 reliance (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH, I). MOLM-13/R and MV4-11/R cells additionally displayed increased HRK sensitivity, implicating BCL-XL engagement, whereas OCI-AML-2/R cells exhibited a predominant MCL-1-dependent shift. Together, these data establish that acquired VEN resistance involves coordinated phenotypic diversification and context-specific rewiring of anti-apoptotic dependencies \u0026mdash; from BCL-2 reliance toward MCL-1- and, in part, BCL-XL-mediated survival.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eIntegrative transcriptomic and metabolomic analysis identifies ADAM15 as a main regulator of venetoclax resistance\u003c/h3\u003e\n\u003cp\u003eRNA sequencing of paired VEN-resistant and parental AML cell lines identified a conserved transcriptional signature comprising 27 upregulated and 222 downregulated genes shared across all three resistant models (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, E).\u003c/p\u003e\n\u003cp\u003eTo assess clinical relevance, we interrogated the BeatAML functional genomics dataset. Differential expression analysis of ex vivo samples stratified by VEN sensitivity (AUC high vs. low) revealed extensive transcriptional reprogramming in VEN-refractory cases (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Analysis of intrinsically resistant OCI-AML-3 cells relative to VEN-sensitive lines identified an additional set of differentially expressed genes associated with primary resistance (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Cross-comparison of these datasets defined a restricted subset of concordantly regulated genes shared across experimental and clinical contexts (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F; Supplementary Tables S1, S2). KEGG pathway enrichment analysis consistently identified cell adhesion\u0026ndash;related programs, with the cell adhesion molecule (CAM) pathway emerging as a recurrent feature across both primary and acquired resistance (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA).\u003c/p\u003e\n\u003cp\u003eGiven emerging evidence linking metabolic adaptation to VEN resistance, we performed untargeted metabolomic profiling of resistant and parental cells, which revealed coordinated alterations in metabolite abundance across all three models (Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB, C). Correlation analysis between differentially expressed genes and resistance-associated metabolites identified ADAM15 as a highly connected node, showing strong correlations with a substantial fraction of altered metabolites across all cell line pairs (|Pearson\u0026apos;s r| \u0026gt; 0.8; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). ADAM15 was consistently upregulated across all experimental and clinical resistance datasets, positioning it as a convergent molecular node at the interface of transcriptional and metabolic reprogramming in VEN-refractory AML.\u003c/p\u003e\n\u003ch3\u003eADAM15 expression correlates with venetoclax resistance and inferior clinical outcome in AML\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eADAM15 expression correlates with venetoclax resistance and inferior clinical outcome in AML\u003c/div\u003e\n\u003cp\u003eHaving identified ADAM15 as a convergently upregulated candidate across multiple VEN-refractory contexts, we next sought to evaluate its clinical relevance in AML. To this end, we integrated ex vivo VEN sensitivity data, matched transcriptomic profiles, and clinical annotation from the BeatAML cohort (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). ADAM15 expression correlated positively with VEN AUC values (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and was significantly elevated in VEN-resistant compared with VEN-sensitive samples (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Kaplan\u0026ndash;Meier analysis of 227 AML patients receiving VEN-based therapy demonstrated that high ADAM15 expression was associated with significantly inferior overall survival (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These data indicate that elevated ADAM15 not only associates with reduced drug sensitivity but also identifies a subgroup of patients with adverse clinical outcomes in the context of VEN-based treatment.\u003c/p\u003e\n\u003cp\u003eTo validate these findings independently, we analyzed an institutional cohort of AML patients stratified by treatment response. ADAM15 mRNA expression was significantly higher in refractory/relapsed cases compared with those achieving remission (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), a pattern corroborated at the protein level by immunoblotting (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, G). Together, these data establish ADAM15 upregulation as a consistent correlate of VEN refractoriness and adverse clinical outcome across independent datasets and patient cohorts.\u003c/p\u003e\n\u003ch3\u003eModulation of ADAM15 alters venetoclax sensitivity and mitochondrial apoptotic threshold in AML\u003c/h3\u003e\n\u003cp\u003eADAM15 was consistently elevated in VEN-resistant AML cells at both the mRNA and protein levels (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). To assess its functional contribution to resistance, we performed reciprocal gain- and loss-of-function studies. Ectopic ADAM15 overexpression in VEN-sensitive parental cells modestly but significantly reduced VEN sensitivity across all three lines (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). Conversely, stable ADAM15 depletion via two independent shRNAs in the corresponding resistant derivatives partially restored VEN sensitivity (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE\u0026ndash;G). These bidirectional findings identify ADAM15 as a functionally relevant modulator of VEN sensitivity in AML.\u003c/p\u003e\n\u003cp\u003eIn addition to drug sensitivity, ADAM15 knockdown also significantly attenuated the migratory and invasive capacities of resistant cells (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, I) and reversed the aberrant cell-cycle response to VEN: whereas control resistant cells exhibited S-phase accumulation upon VEN treatment, ADAM15-depleted cells showed reduced S-phase fraction and increased sub-G₀ apoptotic population (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ).\u003c/p\u003e\n\u003cp\u003eTo investigate the molecular basis of restored sensitivity, we performed RNA sequencing in ADAM15-knockdown and control resistant cells. Although 479 shared differentially expressed genes were identified across the three models (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK), BCL-2 family members did not show a consistent transcriptional pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL), suggesting that ADAM15-mediated resistance operates largely independently of uniform transcriptional reprogramming of core apoptotic machinery. We therefore assessed whether ADAM15 depletion alters mitochondrial apoptotic readiness at the functional level. Dynamic BH3 profiling revealed markedly increased mitochondrial priming in ADAM15-knockdown cells across all three resistant models, as reflected by enhanced BIM-induced depolarization (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eM). In OCI-AML-2/R and MV4-11/R cells, increased priming extended to multiple BH3 peptides and BH3 mimetics, indicating a broad shift toward an apoptosis-permissive state. MOLM-13/R cells showed a similar pattern, with the exception of modestly reduced HRK-induced priming. These data demonstrate that ADAM15 depletion restores VEN sensitivity primarily by increasing mitochondrial apoptotic priming rather than by transcriptional reprogramming of apoptotic regulators.\u003c/p\u003e\n\u003ch3\u003eADAM15 depletion enhances in vivo venetoclax efficacy and prolongs survival in resistant AML models\u003c/h3\u003e\n\u003cp\u003eTo assess whether ADAM15 contributes to VEN resistance in vivo, we established xenograft models using parental OCI-AML-2, resistant OCI-AML-2/R scramble control, and ADAM15-knockdown OCI-AML-2/R cells (Fig. \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, Supplementary Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA). VEN markedly suppressed leukemia progression in parental xenografts but was substantially attenuated in resistant scramble xenografts. ADAM15 depletion restored in vivo VEN responsiveness: mice bearing ADAM15-knockdown resistant cells showed significantly lower peripheral blood hCD45⁺ leukemic chimerism, reduced leukemic infiltration in bone marrow, liver, and spleen, and decreased spleen weight and tumor burden compared with VEN-treated resistant scramble controls (Fig. \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). Histopathological analysis confirmed reduced systemic leukemic infiltration in the ADAM15-knockdown plus VEN group (Fig. \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, Supplementary Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB).\u003c/p\u003e\n\u003cp\u003eLongitudinal monitoring of peripheral hCD45⁺ cells revealed that ADAM15 depletion significantly slowed leukemic expansion under VEN treatment, consistent with delayed disease progression (Fig. \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Notably, ADAM15 depletion alone exerted only a limited effect on leukemic burden, whereas its combination with VEN conferred substantially greater benefit, indicating that ADAM15 knockdown primarily sensitizes resistant cells to VEN rather than broadly suppressing leukemia growth. Mice engrafted with ADAM15-knockdown resistant cells and treated with VEN exhibited significantly prolonged survival compared with VEN-treated scramble controls (Fig. \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e\n\u003cp\u003eTogether, these data demonstrate that ADAM15 depletion restores VEN responsiveness in vivo, suppresses leukemia progression across multiple disease compartments, and prolongs survival in resistant AML xenografts.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eADAM15 drives adaptive survival signaling through HCK-dependent activation of AKT-FOXO3A axis under venetoclax stress\u003c/h2\u003e\n \u003cp\u003eTo identify effectors downstream of ADAM15, we performed protein\u0026ndash;protein interaction network and hub gene analyses using shared differentially expressed genes across the three ADAM15-knockdown resistant models (Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). HCK was consistently downregulated following ADAM15 depletion and predicted to interact with ADAM15, nominating it as a candidate proximal effector (Fig. \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e\n \u003cp\u003eImmunofluorescence analysis revealed increased colocalization of ADAM15 and HCK in VEN-resistant cells following VEN exposure, whereas parental cells displayed substantially weaker overlap (Fig. \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, Supplementary Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA, B). Quantitative colocalization metrics confirmed enhanced spatial coupling between ADAM15 and HCK specifically in resistant cells under therapeutic stress (Fig. \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D). Reciprocal co-immunoprecipitation demonstrated that ADAM15 and HCK formed a detectable complex in resistant OCI-AML-2/R cells following VEN treatment, but not in parental cells or under DMSO conditions (Fig. \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE\u0026ndash;G). These data indicate that ADAM15\u0026ndash;HCK engagement is preferentially induced in resistant cells in response to VEN exposure rather than constitutively maintained.\u003c/p\u003e\n \u003cp\u003eWe next examined downstream signaling consequences of this interaction. Under VEN treatment, resistant cells displayed increased ADAM15 abundance together with enhanced phosphorylation of HCK, PDK1, AKT, GSK3\u0026beta;, and FOXO3A compared with parental cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH, I), consistent with activation of an HCK-PDK1-AKT-FOXO3A survival pathway downstream of ADAM15. VEN-treated resistant cells also showed increased p-BAD and reduced BIM expression alongside sustained MCL-1 elevation, indicating coupled anti-apoptotic remodeling (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH, J, Supplementary Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eE). Under basal DMSO conditions, these signaling changes were less consistent and key phosphorylation events were not robustly detected (Supplementary Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eD), indicating that VEN exposure elicits a more coherent ADAM15-associated survival response than baseline rewiring alone. Nuclear\u0026ndash;cytoplasmic fractionation confirmed enhanced cytoplasmic retention of FOXO3A in resistant cells with a corresponding reduction in the nuclear fraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK, L), consistent with AKT-dependent suppression of FOXO3A pro-apoptotic transcriptional activity.\u003c/p\u003e\n \u003cp\u003eThese results define a VEN stress-induced signaling mechanism in which ADAM15 engages HCK to activate the PDK1\u0026ndash;AKT\u0026ndash;FOXO3A axis, driving adaptive survival signaling and anti-apoptotic remodeling in resistant AML under therapeutic stress.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003ePharmacologic and genetic disruption of the ADAM15-HCK axis re-sensitizes AML to venetoclax\u003c/h3\u003e\n\u003cp\u003eGSEA revealed global suppression of FOXO-associated transcriptional programs in resistant cells (Fig. \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), consistent with enhanced FOXO3A cytoplasmic sequestration in the resistant state. To determine whether this pro-survival signaling is functionally dependent on the ADAM15\u0026ndash;HCK axis, we examined the effects of genetic ADAM15 depletion and pharmacologic HCK inhibition with PP2 in VEN-resistant OCI-AML-2/R cells. VEN treatment induced robust activation of the HCK\u0026ndash;PDK1\u0026ndash;AKT cascade in resistant cells, as evidenced by increased phosphorylation of HCK, PDK1, AKT, GSK3\u0026beta;, and FOXO3A. Both ADAM15 knockdown and PP2 treatment markedly attenuated this signaling cascade under VEN exposure (Fig. \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, Supplementary Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eF), indicating that sustained AKT\u0026ndash;FOXO3A activation requires ADAM15\u0026ndash;HCK engagement. Disruption of this axis also reversed key anti-apoptotic features of the resistant state, reducing BAD phosphorylation and restoring BIM expression (Fig. \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), consistent with reactivation of FOXO3A-dependent pro-apoptotic transcription.\u003c/p\u003e\n\u003cp\u003eFunctionally, whereas resistant cells exhibited limited apoptotic response to VEN monotherapy, both ADAM15 depletion and HCK inhibition significantly enhanced VEN-induced apoptosis, with combined treatment producing a marked increase in apoptotic cell death across multiple resistant models (Fig. \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). These findings establish that the ADAM15\u0026ndash;HCK\u0026ndash;AKT\u0026ndash;FOXO3A axis is required for maintenance of the VEN-resistant state and is therapeutically actionable, with its disruption restoring apoptotic competence and re-sensitizing resistant AML to VEN.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study reveals that venetoclax resistance in AML is not solely a consequence of static anti-apoptotic rewiring but also depends on a dynamic, stress-responsive signaling program orchestrated by ADAM15. Under therapeutic pressure, ADAM15 engages HCK to activate AKT\u0026ndash;FOXO3A signaling, suppressing mitochondrial apoptotic priming and sustaining the resistant state. This mechanism is therapeutically actionable: both genetic ADAM15 depletion and pharmacologic HCK inhibition restore VEN sensitivity in vitro and prolong survival in resistant xenograft models, defining the ADAM15\u0026ndash;HCK axis as a candidate therapeutic target for VEN-refractory AML.\u003c/p\u003e \u003cp\u003eResistance to VEN has been attributed to multiple non-mutually exclusive mechanisms, including compensatory reliance on alternative anti-apoptotic proteins such as MCL1 or BCL-XL\u003csup\u003e7,17,18\u003c/sup\u003e, metabolic adaptation\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, transcriptional and epigenetic reprogramming\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, and activation of pro-survival kinase pathways\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Our findings extend current models of VEN resistance by identifying ADAM15 as a membrane-proximal signaling regulator that links therapeutic stress to adaptive kinase signaling and apoptotic remodeling. Although ADAM15 has been implicated in tumor cell adhesion, angiogenesis, and metastasis in various solid malignancies\u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, its role in hematologic drug resistance has remained unexplored. In this context, our data establish that ADAM15 contributes not only to maintenance of the resistant state but also to associated phenotypic features, including altered cell-cycle dynamics, enhanced migratory and invasive behavior, and reduced mitochondrial apoptotic priming, broadening the biological scope of ADAM15 beyond solid tumor biology.\u003c/p\u003e \u003cp\u003eMechanistically, activation of the ADAM15\u0026ndash;HCK axis imposes a dual restraint on mitochondrial apoptotic competence through downstream AKT signaling. AKT-mediated BAD phosphorylation inactivates this pro-apoptotic BH3-only protein\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, while concurrent FOXO3A phosphorylation and cytoplasmic sequestration suppresses BIM transcription \u0026mdash; consistent with studies in hematologic malignancies linking FOXO3A inactivation to reduced pro-apoptotic transcriptional output\u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Importantly, ADAM15\u0026ndash;HCK engagement is not constitutive but preferentially induced under VEN exposure, distinguishing this mechanism from static anti-apoptotic rewiring and positioning it as a therapeutic stress-adaptive response.\u003c/p\u003e \u003cp\u003eThese findings carry direct translational implications. High ADAM15 expression correlated with reduced ex vivo VEN sensitivity and inferior overall survival in public datasets, observations corroborated in our institutional cohort where ADAM15 was elevated in refractory/relapsed cases \u0026mdash; supporting its potential as a candidate biomarker of VEN response. Therapeutically, the pathway appears pharmacologically tractable. Although ADAM15 currently lacks a well-defined selective small-molecule inhibitor\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, its oncogenic functions appear to depend mainly on scaffold-mediated signaling rather than on its metalloproteinase activity\u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. This scaffold-dependent mode of action suggests therapeutic strategies targeting protein degradation (e.g., PROTACs) or disruption of the ADAM15\u0026ndash;HCK interface. More immediately, co-treatment with the SFK inhibitor PP2 and VEN markedly increased apoptosis in resistant cells, recapitulating genetic ADAM15 depletion. Given that SFK inhibitors (SFKi) such as dasatinib are already clinically deployed in hematologic malignancies, and that emerging evidence supports SFKi-containing regimens enhancing BCL-2-directed therapy\u003csup\u003e\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, our findings provide a mechanistic rationale for SFK-based combination strategies in VEN-refractory AML.\u003c/p\u003e \u003cp\u003eSeveral limitations warrant acknowledgment. First, the downstream transcriptional programs through which FOXO3A executes this adaptive survival state remain incompletely defined and will require integrative chromatin profiling. Second, the functional relationship between this signaling axis and the metabolic reprogramming observed in resistant cells \u0026mdash; whether downstream effector or parallel adaptation \u0026mdash; also remains to be dissected. Finally, validation in patient-derived xenograft models will be necessary to assess translational feasibility more rigorously.\u003c/p\u003e \u003cp\u003eIn conclusion, this study identifies ADAM15 as a convergent driver of VEN resistance in AML, operating through stress-induced HCK\u0026ndash;PDK1\u0026ndash;AKT\u0026ndash;FOXO3A signaling to suppress mitochondrial apoptotic priming. These findings expand the molecular framework of BCL-2 inhibitor refractoriness and provide a rationale for therapeutic strategies targeting the ADAM15\u0026ndash;HCK axis to restore VEN sensitivity.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eDetailed information on all shRNA sequences, primer sequences, BH3 peptides, and antibodies is provided in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Detailed experimental protocols are provided in Supplementary Methods.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePatient samples\u003c/h2\u003e \u003cp\u003ePeripheral blood or bone marrow mononuclear cells were isolated by Ficoll density gradient centrifugation from AML patients treated at Guangdong General Hospital. Samples were classified into a VEN-remission group (patients achieving CR/CRi following venetoclax-based therapy) and a VEN-refractory/relapsed group (patients failing to achieve CR/CRi or relapsing after initial response). Clinical characteristics are summarized in Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. Written informed consent was obtained in accordance with the Declaration of Helsinki. The study was approved by the Ethics Committee of Guangdong General Hospital (approval no. KY-Z-2020-551-02).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and establishment of venetoclax-resistant cells\u003c/h2\u003e \u003cp\u003eHuman AML cell lines (MOLM-13, OCI-AML-2, OCI-AML-3, MV4-11, KG1α, THP-1) and HEK293T cells were maintained under standard conditions (Supplementary Methods). VEN-resistant subclones of OCI-AML-2, MV4-11, and MOLM-13 were generated through continuous exposure to stepwise increasing concentrations of ABT-199, beginning at the IC₅₀ and escalating 1.5\u0026ndash;2-fold per cycle until cells proliferated stably at concentrations exceeding 10-fold the initial IC₅₀. Resistant cells were thereafter maintained in ABT-199-containing medium. All cell lines were authenticated by STR profiling before and after resistance induction and routinely tested for Mycoplasma contamination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCell viability assays\u003c/h2\u003e \u003cp\u003eCell viability was assessed by CCK-8 assay following 48-h treatment with ABT-199 or vehicle control. IC₅₀ values were determined by four-parameter nonlinear regression. All experiments were performed with at least three biological replicates in technical triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMigration and invasion assays\u003c/h2\u003e \u003cp\u003eMigration and invasion were assessed using Transwell inserts with or without Matrigel coating. Cells were seeded in serum-free medium in the upper chamber, allowed to migrate toward serum-containing medium for 24 h, and quantified by flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eApoptosis was assessed by Annexin V/PI staining following ABT-199 treatment. Cell cycle distribution was analyzed by PI/RNase staining and modeled using ModFit LT. Human cell engraftment in xenograft experiments was quantified as the percentage of hCD45⁺mCD45⁻ cells among total viable CD45⁺ leukocytes. All flow cytometric analyses were performed on a Cytek Aurora Evo flow cytometer and analyzed using FlowJo (v10.8.1). Detailed staining protocols, gating strategies, and quality control criteria are provided in Supplementary Methods.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eLentiviral-mediated gene knockdown and overexpression\u003c/h2\u003e \u003cp\u003eADAM15 overexpression and shRNA knockdown constructs were delivered by lentiviral transduction into parental and resistant AML cell lines, respectively. Transduced cells were selected with puromycin, and knockdown/overexpression efficiency was verified by RT-qPCR and immunoblotting. shRNA sequences are provided in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e; vector and transduction details are provided in Supplementary Methods.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eBH3 profiling\u003c/h2\u003e \u003cp\u003eDynamic BH3 profiling was performed on digitonin-permeabilized cells using a panel of BH3 peptides (BIM, BID, HRK, NOXA, BAD, PUMA) and BH3 mimetics (ABT-199, S63845, WHI-539). Mitochondrial depolarization was monitored kinetically by JC-1 fluorescence over 2 h, and priming was calculated relative to CCCP-induced complete depolarization. Peptide sequences, concentrations, and detailed protocols are provided in Supplementary Methods.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and real-time q-PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using TRIzol, reverse-transcribed, and analyzed by SYBR Green-based qPCR on an ABI 7500 system. Relative expression was normalized to GAPDH using the 2⁻\u003csup\u003eΔΔCt\u003c/sup\u003e method. Each biological replicate was measured in technical triplicate where indicated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eRNA sequencing\u003c/h2\u003e \u003cp\u003ePoly(A)-selected RNA libraries were constructed and sequenced on an Illumina NovaSeq 6000 platform in paired-end 150 bp mode. Reads were aligned to the reference genome using HISAT2, quantified using StringTie, and differential expression analysis was performed using DESeq2 (|log₂FC| \u0026ge; 1, adjusted P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Raw sequencing data have been deposited in the NCBI Gene Expression Omnibus (GEO) under accession number GSEXXXXXX. (Raw sequencing data have been submitted to the GEO and are currently under processing; the accession number will be inserted prior to publication.).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eUntargeted metabolomics\u003c/h2\u003e \u003cp\u003eParental and VEN-resistant cell line pairs (MOLM-13, OCI-AML-2, MV4-11) prepared in parallel with RNA-seq samples were subjected to untargeted metabolomic profiling. Metabolites were identified by accurate mass, retention time, and MS/MS fragmentation against reference databases, with differentially abundant metabolites defined by VIP\u0026thinsp;\u0026gt;\u0026thinsp;1.0.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eCo-immunoprecipitation\u003c/h2\u003e \u003cp\u003eTo assess the ADAM15\u0026ndash;HCK interaction under therapeutic stress, cells were treated with whether ABT-199 or DMSO for 12 h and subjected to reciprocal co-immunoprecipitation using crosslink-based IP with antibodies against ADAM15 and HCK, with corresponding isotype controls. Bound proteins were analyzed by immunoblotting.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eImmunoblotting\u003c/h2\u003e \u003cp\u003eWhole-cell lysates or subcellular fractions were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with the indicated primary and HRP-conjugated secondary antibodies. For HCK inhibition studies, cells were pretreated with PP2 prior to ABT-199 exposure. Band intensities were quantified using ImageJ. Antibody details are provided in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e; full protocols including treatment conditions, lysis buffers, and fractionation procedures are provided in Supplementary Methods.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining and confocal microscopy\u003c/h2\u003e \u003cp\u003eCells were treated with ABT-199 or vehicle for 2 h, fixed, permeabilized with digitonin, and stained with primary antibodies followed by Alexa Fluor-conjugated secondary antibodies. Nuclei were counterstained with DAPI. Images were acquired by confocal microscopy, and colocalization was quantified using ImageJ.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eIn vivo xenograft assays\u003c/h2\u003e \u003cp\u003eFemale NOD/SCID mice (6 weeks) were intravenously injected with 4 \u0026times; 10⁶ cells from one of the following groups: parental OCI-AML-2, OCI-AML-2/R, OCI-AML-2/R-shADAM15, OCI-AML-2/R-scramble, or PBS alone (cell-free control). Engraftment was confirmed at day 7 by peripheral blood hCD45 flow cytometry and Wright-Giemsa morphology, with successful engraftment defined as \u0026ge;\u0026thinsp;1% hCD45⁺ cells. Engrafted mice were randomized (n\u0026thinsp;\u0026ge;\u0026thinsp;6 per group) to receive venetoclax (50 mg/kg, oral gavage, QD) or vehicle beginning on day 7. Disease progression was monitored weekly by peripheral blood hCD45/mCD45 chimerism. Mice were euthanized upon reaching predefined humane endpoints, and tissues were collected for flow cytometry, histology, immunohistochemistry, and molecular analyses. All procedures were approved by the IACUC (approval no. G2025074) and conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eBeatAML Cohort Analysis\u003c/h2\u003e \u003cp\u003eClinical, transcriptomic, and drug sensitivity data were obtained from the BeatAML database\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Patients were stratified into venetoclax-sensitive (Q1) and venetoclax-resistant (Q4) groups by venetoclax AUC quartiles. ADAM15 expression was compared between groups and correlated with venetoclax sensitivity. Patients were further dichotomized into ADAM15-high and ADAM15-low groups using the optimal cutoff, and overall survival was analyzed by Kaplan-Meier method with log-rank testing.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eExperiments were performed with at least three independent biological replicates, and data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD unless otherwise stated. Comparisons between two groups were made using unpaired two-tailed Student's t-test; multiple-group comparisons used one-way ANOVA with Tukey's post hoc test. IC₅₀ values were determined by four-parameter nonlinear regression. Survival was analyzed by Kaplan-Meier method with log-rank test. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. All analyses were performed using GraphPad Prism (v9.5) or R (v4.3.1).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eRNA sequencing data have been submitted to the NCBI Gene Expression Omnibus (GEO) and are currently under processing; the accession number will be inserted prior to publication. All other data supporting the findings of this study are available within the article and its supplementary files, or from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Guangdong Basic and Applied Basic Research Foundation (2024B1515020054), the National Natural Science Foundation of China (82270161, 82500188), the Science and Technology Planning Project of Guangdong Province (2023B1111050004), the Project of Administration of Traditional Chinese Medicine of Guangdong Province (20242002), and the Medical Scientific Research Foundation of Guangdong Province (B2025364).\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eKFL and PLL conceived and designed the study. KFL, LJZ and MZX performed the experiments and analyzed the data. KFL wrote the manuscript. MZX, SXG, YLW, MW, PLL and RHX reviewed and edited the manuscript. XH, MML, PW, XMC and YTL provided the clinical samples/datasets. XD, JYW and PLL supervised and acquired funding for this study. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eEthics declarations\u003c/p\u003e\n\u003cp\u003eThis study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Guangdong General Hospital (approval no. KY-Z-2020-551-02). All procedures involving animal subjects were approved by the Institutional Animal Care and Use Committee (IACUC; approval no. G2025074).\u003c/p\u003e\n\u003cp\u003eConflit of interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eShort NJ, Daver N, Dinardo CD, Kadia T, Nasr LF, Macaron W \u003cem\u003eet al.\u003c/em\u003e Azacitidine, venetoclax, and gilteritinib in newly diagnosed and relapsed or refractory FLT3-mutated AML. \u003cem\u003eJ Clin Oncol Off J Am Soc Clin Oncol\u003c/em\u003e 2024; 42: 1499\u0026ndash;1508.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei AH, Panayiotidis P, Montesinos P, Laribi K, Ivanov V, Kim I \u003cem\u003eet al.\u003c/em\u003e Long-term follow-up of VIALE-C in patients with untreated AML ineligible for intensive chemotherapy. \u003cem\u003eBlood\u003c/em\u003e 2022; 140: 2754\u0026ndash;2756.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei AH, Loo S, Daver N. How I treat patients with AML using azacitidine and venetoclax. \u003cem\u003eBlood\u003c/em\u003e 2025; 145: 1237\u0026ndash;1250.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKonopleva M, Thirman MJ, Pratz KW, Garcia JS, Recher C, Pullarkat V \u003cem\u003eet al.\u003c/em\u003e Impact of FLT3 mutation on outcomes after venetoclax and azacitidine for patients with treatment-na\u0026iuml;ve acute myeloid leukemia. \u003cem\u003eClin Cancer Res Off J Am Assoc Cancer Res\u003c/em\u003e 2022; 28: 2744\u0026ndash;2752.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePratz KW, Jonas BA, Pullarkat V, Thirman MJ, Garcia JS, D\u0026ouml;hner H \u003cem\u003eet al.\u003c/em\u003e Long-term follow-up of VIALE-a: Venetoclax and azacitidine in chemotherapy-ineligible untreated acute myeloid leukemia. \u003cem\u003eAm J Hematol\u003c/em\u003e 2024; 99: 615\u0026ndash;624.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePei S, Pollyea DA, Gustafson A, Stevens BM, Minhajuddin M, Fu R \u003cem\u003eet al.\u003c/em\u003e Monocytic subclones confer resistance to venetoclax-based therapy in patients with acute myeloid leukemia. \u003cem\u003eCancer Discov\u003c/em\u003e 2020; 10: 536\u0026ndash;551.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Q, Riley-Gillis B, Han L, Jia Y, Lodi A, Zhang H \u003cem\u003eet al.\u003c/em\u003e Activation of RAS/MAPK pathway confers MCL-1 mediated acquired resistance to BCL-2 inhibitor venetoclax in acute myeloid leukemia. \u003cem\u003eSignal Transduct Target Ther\u003c/em\u003e 2022; 7: 51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGui\u0026egrave;ze R, Liu VM, Rosebrock D, Jourdain AA, Hern\u0026aacute;ndez-S\u0026aacute;nchez M, Martinez Zurita A \u003cem\u003eet al.\u003c/em\u003e Mitochondrial reprogramming underlies resistance to BCL-2 inhibition in lymphoid malignancies. \u003cem\u003eCancer Cell\u003c/em\u003e 2019; 36: 369\u0026ndash;384.e13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMontero J, Letai A. Why do BCL-2 inhibitors work and where should we use them in the clinic? \u003cem\u003eCell Death Differ\u003c/em\u003e 2018; 25: 56\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou J, Wang A, Cai T, Li Y, Du W, Zhang Y \u003cem\u003eet al.\u003c/em\u003e Integrin α3/α6 and αV are implicated in ADAM15-activated FAK and EGFR signalling pathway individually and promote non-small-cell lung cancer progression. \u003cem\u003eCell Death Dis\u003c/em\u003e 2022; 13: 486.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCaba\u0026ntilde;as C, Rossi E, Bartolom\u0026eacute; RA, Doberstein K, Altevogt P, Casal JI \u003cem\u003eet al.\u003c/em\u003e The emerging role of human transmembrane RGD-based counter-receptors of integrins in health and disease. \u003cem\u003eCell Mol Biol Lett\u003c/em\u003e 2025; 30: 110.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEvers M, St\u0026uuml;hmer T, Schreder M, Steinbrunn T, Rudelius M, Jundt F \u003cem\u003eet al.\u003c/em\u003e Association of ADAM family members with proliferation signaling and disease progression in multiple myeloma. \u003cem\u003eBlood Cancer J\u003c/em\u003e 2024; 14: 156.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCiechomska IA, Gielniewski B, Wojtas B, Kaminska B, Mieczkowski J. EGFR/FOXO3a/BIM signaling pathway determines chemosensitivity of BMP4-differentiated glioma stem cells to temozolomide. \u003cem\u003eExp Mol Med\u003c/em\u003e 2020; 52: 1326\u0026ndash;1340.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Y, Wang Y, Li X, Jia Y, Wang J, Ao X. FOXO3a in cancer drug resistance. \u003cem\u003eCancer Lett\u003c/em\u003e 2022; 540: 215724.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGong K, Xu B, Gong L, Zhong M, Han C, Liu Y \u003cem\u003eet al.\u003c/em\u003e ITGA2 mediates the resistance of hepatocellular carcinoma to lenvatinib by activating the AKT/FOXO3A signaling pathway. \u003cem\u003eCancers\u003c/em\u003e 2025; 17. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/cancers17172846\u003c/span\u003e\u003cspan address=\"10.3390/cancers17172846\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa J, Zhao S, Qiao X, Knight T, Edwards H, Polin L \u003cem\u003eet al.\u003c/em\u003e Inhibition of bcl-2 synergistically enhances the antileukemic activity of midostaurin and gilteritinib in preclinical models of FLT3-mutated acute myeloid leukemia. \u003cem\u003eClin Cancer Res Off J Am Assoc Cancer Res\u003c/em\u003e 2019; 25: 6815\u0026ndash;6826.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuusanm\u0026auml;ki H, Dufva O, V\u0026auml;h\u0026auml;-Koskela M, Lepp\u0026auml; A-M, Huuhtanen J, V\u0026auml;nttinen I \u003cem\u003eet al.\u003c/em\u003e Erythroid/megakaryocytic differentiation confers BCL-XL dependency and venetoclax resistance in acute myeloid leukemia. \u003cem\u003eBlood\u003c/em\u003e 2023; 141: 1610\u0026ndash;1625.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePan R, Hogdal LJ, Benito JM, Bucci D, Han L, Borthakur G \u003cem\u003eet al.\u003c/em\u003e Selective BCL-2 inhibition by ABT-199 causes on-target cell death in acute myeloid leukemia. \u003cem\u003eCancer Discov\u003c/em\u003e 2014; 4: 362\u0026ndash;375.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen X, Glytsou C, Zhou H, Narang S, Reyna DE, Lopez A \u003cem\u003eet al.\u003c/em\u003e Targeting mitochondrial structure sensitizes acute myeloid leukemia to venetoclax treatment. \u003cem\u003eCancer Discov\u003c/em\u003e 2019; 9: 890\u0026ndash;909.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoca-Portoles A, Rodriguez-Blanco G, Sumpton D, Cloix C, Mullin M, Mackay GM \u003cem\u003eet al.\u003c/em\u003e Venetoclax causes metabolic reprogramming independent of BCL-2 inhibition. \u003cem\u003eCell Death Dis\u003c/em\u003e 2020; 11: 616.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThomalla D, Beckmann L, Grimm C, Oliverio M, Meder L, Herling CD \u003cem\u003eet al.\u003c/em\u003e Deregulation and epigenetic modification of BCL2-family genes cause resistance to venetoclax in hematologic malignancies. \u003cem\u003eBlood\u003c/em\u003e 2022; 140: 2113\u0026ndash;2126.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSatta T, Li L, Chalasani SL, Hu X, Nkwocha J, Sharma K \u003cem\u003eet al.\u003c/em\u003e Dual mTORC1/2 inhibition synergistically enhances AML cell death in combination with the BCL2 antagonist venetoclax. \u003cem\u003eClin Cancer Res Off J Am Assoc Cancer Res\u003c/em\u003e 2023; 29: 1332\u0026ndash;1343.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuhachek-Muggy S, Qi Y, Wise R, Alyahya L, Li H, Hodge J \u003cem\u003eet al.\u003c/em\u003e Metalloprotease-disintegrin ADAM12 actively promotes the stem cell-like phenotype in claudin-low breast cancer. \u003cem\u003eMol Cancer\u003c/em\u003e 2017; 16: 32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDatta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y \u003cem\u003eet al.\u003c/em\u003e Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. \u003cem\u003eCell\u003c/em\u003e 1997; 91: 231\u0026ndash;241.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGilley J, Coffer PJ, Ham J. FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons. \u003cem\u003eJ Cell Biol\u003c/em\u003e 2003; 162: 613\u0026ndash;622.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDijkers PF, Medema RH, Lammers JW, Koenderman L, Coffer PJ. Expression of the pro-apoptotic bcl-2 family member bim is regulated by the forkhead transcription factor FKHR-L1. \u003cem\u003eCurr Biol CB\u003c/em\u003e 2000; 10: 1201\u0026ndash;1204.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosich L, Saborit-Villarroya I, L\u0026oacute;pez-Guerra M, Xargay-Torrent S, Montraveta A, Aymerich M \u003cem\u003eet al.\u003c/em\u003e The phosphatidylinositol-3-kinase inhibitor NVP-BKM120 overcomes resistance signals derived from microenvironment by regulating the akt/FoxO3a/bim axis in chronic lymphocytic leukemia cells. \u003cem\u003eHaematologica\u003c/em\u003e 2013; 98: 1739\u0026ndash;1747.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChung Y-L, Pan C-H, Wang CC-N, Hsu K-C, Sheu M-J, Chen H-F \u003cem\u003eet al.\u003c/em\u003e Methyl protodioscin, a steroidal saponin, inhibits neointima formation in vitro and in vivo. \u003cem\u003eJ Nat Prod\u003c/em\u003e 2016; 79: 1635\u0026ndash;1644.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoujalled DM, Hanna DT, Hediyeh-Zadeh S, Pomilio G, Brown L, Litalien V \u003cem\u003eet al.\u003c/em\u003e Cotargeting BCL-2 and MCL-1 in high-risk B-ALL. \u003cem\u003eBlood Adv\u003c/em\u003e 2020; 4: 2762\u0026ndash;2767.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarciaz S, Montersino C, Bourgoin M, Jacquel A, Castellano R, Guille A \u003cem\u003eet al.\u003c/em\u003e Dasatinib overcomes AML cells resistant to BCL2 inhibition by degrading MCL1. \u003cem\u003eBr J Haematol\u003c/em\u003e 2025; 207: 381\u0026ndash;386.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGocho Y, Liu J, Hu J, Yang W, Dharia NV, Zhang J \u003cem\u003eet al.\u003c/em\u003e Network-based systems pharmacology reveals heterogeneity in LCK and BCL2 signaling and therapeutic sensitivity of T-cell acute lymphoblastic leukemia. \u003cem\u003eNat Cancer\u003c/em\u003e 2021; 2: 284\u0026ndash;299.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTyner JW, Tognon CE, Bottomly D, Wilmot B, Kurtz SE, Savage SL \u003cem\u003eet al.\u003c/em\u003e Functional genomic landscape of acute myeloid leukaemia. \u003cem\u003eNature\u003c/em\u003e 2018; 562: 526\u0026ndash;531.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"leukemia","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"leu","sideBox":"Learn more about [Leukemia](http://www.nature.com/leu/)","snPcode":"41375","submissionUrl":"https://mts-leu.nature.com/cgi-bin/main.plex","title":"Leukemia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9498017/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9498017/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eVenetoclax-based regimens have improved outcomes in acute myeloid leukemia (AML), but resistance frequently emerges and limits durable benefit. The upstream signaling mechanisms linking therapeutic stress to apoptotic evasion remain poorly defined. Here, we identify ADAM15 as a previously unrecognized regulator of venetoclax resistance in AML. Integrative multi-omics analyses across experimental models and clinical cohorts reveal that ADAM15 is consistently upregulated in venetoclax refractory AML and is associated with diminished drug sensitivity and adverse clinical outcomes. Ectopic expression of ADAM15 in parental sensitive cells confer venetoclax resistance, while genetic depletion of ADAM15 in resistant counterparts restores drug sensitivity and enhances mitochondrial apoptotic priming in vitro, and suppresses leukemia progression in resistant xenograft models. Mechanistically, ADAM15 engages HCK under venetoclax exposure to activate an AKT-FOXO3A survival program, thereby sustaining resistance. Collectively, these findings establish ADAM15 as a convergent and therapeutically actionable vulnerability in venetoclax-refractory AML and provide a translational rationale for targeting this axis to overcome resistance.\u003c/p\u003e","manuscriptTitle":"ADAM15 drives venetoclax resistance via HCK–AKT–FOXO3A signaling in AML","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-18 07:02:49","doi":"10.21203/rs.3.rs-9498017/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-05-08T23:50:30+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-05-07T22:06:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-23T13:18:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-23T13:11:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Leukemia","date":"2026-04-22T14:56:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"leukemia","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"leu","sideBox":"Learn more about [Leukemia](http://www.nature.com/leu/)","snPcode":"41375","submissionUrl":"https://mts-leu.nature.com/cgi-bin/main.plex","title":"Leukemia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"fecabbaa-20f6-46fb-acef-88edf42d11d6","owner":[],"postedDate":"May 18th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-05-08T23:50:30+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"2","date":"2026-05-07T22:06:06+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":67743668,"name":"Health sciences/Medical research/Translational research"},{"id":67743669,"name":"Health sciences/Diseases/Haematological diseases/Haematological cancer/Leukaemia/Acute myeloid leukaemia"},{"id":67743670,"name":"Health sciences/Medical research/Preclinical research"}],"tags":[],"updatedAt":"2026-05-18T07:02:49+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-18 07:02:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9498017","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9498017","identity":"rs-9498017","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}