Fusion-specific regulation of polyamine synthesis sensitizes to BCL-XL inhibition in TCF3::PBX1+ B-ALL Free

Fusion transcription factors (TF) are hallmark oncogenic drivers in pediatric B cell acute lymphoblastic leukemia (B-ALL), contributing to leukemia initiation and transformation. Despite advances in risk-adapted therapies guided by clinical and molecular features, genetic heterogeneity and variable outcomes underscore the need to uncover driver-specific vulnerabilities to expand personalized treatment strategies, particularly in the relapsed/refractory context. Here, we apply a systematic approach to identify rational combination strategies informed by integrating identification of driver-specific downstream targets of the fusion TF TCF3::PBX1 – a common driver in pediatric B-ALL – with an unbiased high-throughput drug combination screen.

To dissect fusion-specific druggable genes we knocked down (KD) TCF3::PBX1 by short hairpin RNA (shRNA) in the 697 cell line and filtered for transcriptional targets with available approved and clinical-phase candidate drugs. Among top fusion-associated genes we identified ornithine decarboxylase (ODC1), the rate-limiting enzyme of synthesizing polyamines, small polycations with high affinity for nucleic acids controlling key cellular processes like transcription, translation, DNA replication and cell cycle progression. Public datasets confirmed highest mRNA expression of ODC1 in TCF3::PBX1+ patient samples across B-ALL subtypes (St. Jude) and selective dependency on ODC1 in TCF3::PBX1+ cell lines (DepMap) reinforcing its role as a specific fusion-driven vulnerability. This subtype-specific overexpression of ODC1 led us to investigate its regulation, and public ChIP-seq data revealed binding peaks of TCF3::PBX1 at the ODC1 promoter supporting its role as a novel regulator alongside MYC. Genetic interference by shRNA-mediated ODC1 KD and pharmacological inhibition by Difluoromethylornithine (DFMO) reduced intracellular polyamine levels of putrescine, spermidine and spermine and demonstrated cytostatic effects with decreased proliferation with a reduction of cells in S-Phase in TCF3::PBX1+ cell lines. In patient-derived xenograft (PDX) models, ex vivo sensitivity to DFMO was highest in TCF3::PBX1+ PDX compared to other subtypes. Notably, PDXs from TCF3::PBX1+ relapses exhibited sensitivity levels comparable to those observed in TCF3::PBX1+ PDX with favorable outcomes. Transcriptomic and proteomic analysis of DFMO-treated 697 cells uncovered changes in gene and protein expression associated with amino acid and polyamine transport, mitochondrial metabolism, global translation and cell cycle. Strikingly, treatment with DFMO induced regulation of key B cell identity TF and B cell receptor signaling components, alongside an enrichment of transcriptional signatures indicative of more mature B cell states.

To uncover synthetic lethal vulnerabilities of this induced rewired cell state we performed an unbiased high-throughput combination screen (n = 155 drugs) in 697 cells with or without ODC1 KD. Among known synergistic targets like AMXT-1501, a polyamine transport inhibitor, BCL-XL inhibitors emerged as potent synergistic partners to genetic and pharmacological ODC1 inhibition, further validated in PDX models ex vivo. To evaluate this combination in vivo, we employed a dual-targeting strategy against polyamine metabolism using DFMO to inhibit synthesis and AMXT-1501 to block uptake, as ex vivo data revealed pronounced metabolic plasticity and compensatory upregulation of polyamine transporters following DFMO monotherapy. In vivo combination of DFMO + polyamine-transport inhibitor AMXT-1501 and BCL-XL PROTAC DT2216 in TCF3::PBX1+ PDX models demonstrated promising antileukemic efficacy seen by reduced leukemic burden in spleen and bone marrow at the end of treatment.

Collectively, our findings identify the fusion TF TCF3::PBX1 as a previously unrecognized positive regulator of polyamine synthesis, acting via ODC1 upregulation, positioning ODC1 as a promising target in this subtype. We further demonstrated that targeting polyamine synthesis induces transcriptional changes in essential B-cell developmental and metabolic programs, uncovering opportunities to target synthetic lethal principles through BCL-XL. The recent approval of DFMO for neuroblastoma maintenance therapy highlights its translational potential not only in TCF3::PBX1+ B-ALL but warrants exploration of this rational combination in other MYC-driven and/or polyamine-addicted B-ALL, especially in the relapsed/refractory context.