Targeting CPNE8 suppresses HOXA9-dependent AML progression and overcomes chemotherapy resistance Free

Background Acute myeloid leukemia (AML) remains a challenge in treatment and mechanisms understanding in disease progression and therapeutic resistance. Through large-scale transcriptomic profiling integrated with 10 machine learning algorithms, we identified CPNE8, a calcium-dependent phospholipid-binding protein which is involved in signal transduction, is significantly upregulated in HOXA-driven AML subtypes, whose function and regulatory mechanisms in leukemia warrants further investigation.

Methods We integrated clinical and transcriptomic profiles from a cohort of 655 AML patients to develop a machine learning–based prognostic model, with external validation performed in the TCGA-LAML (n = 151) and GSE16432 (n = 436) datasets. CPNE8 expression across hematopoietic and immune cell compartments was profiled using single-cell RNA sequencing (GSE116256). Functional assays following CPNE8 knockdown were performed to evaluate cell viability, apoptosis, cell cycle dynamics, and leukemogenic potential in vivo. CPNE8-interacting proteins were identified via Co-immunoprecipitation (Co-IP) and Mass Spectrum (MS), with downstream signaling examined by immunoblotting. CUT&Tag profiling and dual-luciferase reporter assays were used to elucidate transcriptional regulation. OCR and ECAR were measured to assess cellular mitochondrial respiration and glycolytic capacity. Chemoresistance was assessed through compound screening and CellTiter-Glo assays, and the therapeutic efficacy of Menin inhibition was tested both in vitro and in vivo using a CPNE8-high OCI-AML3 xenograft model.

ResultsCPNE8 was markedly upregulated in HOXA9-driven AML and associated with poor prognosis and chemoresistance. Patients with high CPNE8 expression exhibited significantly lower complete remission (CR) rates following two cycles of standard induction chemotherapy compared to CPNE8-low patients (p < 0.001), with pronounced resistance to conventional “3+7” anthracycline-based regimens. CPNE8 knockdown inhibited leukemic cell proliferation, induced apoptosis, and caused G0/G1 cell cycle arrest. Mechanically, CPNE8 physically interacted with SmgGDS, as identified by Co-IP–mass spectrometry and further validated by Co-IP assays and immunofluorescence co-localization，thereby activating the Rap1 GTPase module and driving downstream PI3K-AKT and MAPK (JNK/p38) signaling. To further assess the role of CPNE8 in drug resistance, ex vivo drug sensitivity assays were conducted using PBMCs from 107 AML patients. CPNE8-high PBMCs showed significantly elevated IC50 values across a range of clinically relevant chemotherapeutics, including idarubicin, homoharringtonine, fludarabine, azacitidine, venetoclax, aclarubicin, all-trans retinoic acid (ATRA), and mitoxantrone. Additionally, CPNE8 depletion elevated intracellular and mitochondrial ROS, reduced mitochondrial membrane potential, and impaired both glycolysis and oxidative phosphorylation, thereby sensitizing AML cells to chemotherapy and enhancing their vulnerability to T cell–mediated cytotoxicity. To delineate the upstream regulatory network, CUT&Tag profiling identified HOXA9 as a direct transcriptional activator of CPNE8, positioning CPNE8 as a critical effector of HOXA9-driven leukemogenesis. To therapeutically target the HOXA9–CPNE8 axis in CPNE8-high AML, we evaluated Menin inhibition as a strategy to disrupt the HOXA9 transcriptional program. Treatment with a Menin inhibitor significantly reduced CPNE8 expression in vitro. Importantly, both Menin inhibition and CPNE8 knockdown prolonged survival in CPNE8-high AML xenograft models, underscoring their therapeutic potential.

ConclusionCPNE8 functions as a critical downstream effector of HOXA9, driving AML progression and therapy resistance through activation of Rap1 signaling, reprogramming of mitochondrial metabolism, and reshaping of the immune microenvironment. Blocking the HOXA9–CPNE8–Rap1 pathway, especially with Menin inhibitors, provides a rationale for stratified treatment strategies in CPNE8-high AML.