Inhibition of nicotinamide metabolism by the novel NAMPT inhibitor OT-82 potentiates venetoclax in paediatric and adult acute myeloid leukaemia models

Abstract Introduction While the survival rate of paediatric acute lymphoblastic leukaemia (ALL) has increased up to 90%, the prognosis for both paediatric and adult acute myeloid leukaemia (AML) remains poor, with adult 5-year survival rates typically around 25–30% and paediatric outcomes showing minimal improvement over the past five decades. Venetoclax, a selective inhibitor of the pro-survival protein BCL-2, has revolutionised the treatment of adult AML in combination with azacitidine and is currently under investigation in children. However, with reports of inherent and acquired resistance to venetoclax on the rise, it is essential to understand and identify mitigation strategies for venetoclax resistance in adult and paediatric AML. Venetoclax is remarkably more effective in de novo AML than in the relapsed/refractory setting and leukaemic stem cells (LSCs) from relapsed/refractory AML patients have been proposed to exhibit elevated nicotinamide metabolism and increased dependence on Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in nicotinamide adenine dinucleotide (NAD⁺) biosynthesis, as a means to evade venetoclax treatment. The aim of this study was to establish whether the altered dependency of relapsed/refractory AML LSCs on nicotinamide metabolism can be therapeutically exploited to potentiate venetoclax, particularly through the use of OT-82, a clinical-stage NAMPT inhibitor. MethodsVenetoclax was combined with a newly developed NAMPT inhibitor OT-82, and the efficacy of the drug combination was evaluated in AML cell lines in vitro and primary AML bone marrow patient samples ex vivo by resazurin reduction-based viability and synergy assays, live/dead and apoptosis assays. Paediatric venetoclax-resistant AML patient derived xenograft (PDX) models (n=7), were established in immunocompromised MISTRG mice expressing humanised M-CSF, IL3/GM-CSF, SIRPα, TPO in the Rag2-/-, IL2ry-/-background.MISTRG mice were treated with OT-82 or venetoclax monotherapy, or the combination in a Single Mouse Trial (SMT) design to assess therapeutic benefit. In this approach, each PDX model is represented by a single mouse per treatment arm, enabling broad coverage of genetically diverse leukaemias. ResultsNAMPT inhibition by OT-82 killed a molecularly diverse panel of AML cell lines in vitro and primary patient samples from de novo and relapsed/refractory AML patients ex vivo with nanomolar IC50s. The drug induced caspase-dependent apoptosis in leukaemia cells while sparing CD34+ cells from healthy donors. Strong synergy was observed between OT-82 and venetoclax in venetoclax-resistant AML cell lines. Comparable synergy was also achieved with the NAMPT inhibitor KPT9274 in combination with venetoclax. In vivo, OT-82 potentiated venetoclax and venetoclax/azacitidine in a venetoclax-resistant paediatric AML cell line xenograft model, significantly extending survival of mice treated with OT-82/venetoclax or OT-82/venetoclax/azacitidine in comparison to the single agents or venetoclax/azacitidine. Notably, paediatric venetoclax-resistant PDXs were highly sensitive to OT-82, with an enhanced response observed when combined with venetoclax. SMT profiling in adult AML PDXs is currently underway to complement the SMT studies in paediatric AML PDX studies and identify molecular predictors of OT-82 responsiveness at both the genomic and transcriptomic levels. ConclusionOT-82 potentiates venetoclax in venetoclax-resistant AML in vitro and in vivo, including models of paediatric AML, a particularly high-risk and underexplored population. Ongoing SMT-guided molecular profiling studies in adult AML PDX models will further elucidate predictive markers of response. These findings support the rationale for advancing OT-82 plus venetoclax as a novel, metabolism-targeted combination therapy in adult and paediatric AML. This strategy offers a translationally actionable approach to overcome resistance and improve outcomes for AML patients, particularly those with venetoclax-resistant disease who currently face dismal survival rates. Collectively, this work highlights NAMPT inhibition as a promising avenue to enhance venetoclax efficacy and paves the way for clinical translation in both frontline and relapsed/refractory settings.