Efficacy of the JAK2/FLT3 inhibitor pacritinib in NPM1 mutated Acute Myeloid Leukemia Free

Background Acute myeloid leukemia (AML) with mutation in NPM1 (NPM1mut) represents a third ofadult AML cases (Falini et al., 2020). When the mutation co-occurs with FLT3 and/or DNMT3A mutations, the prognosis and response to treatment is less favorable (Falini et al., 2020; Chin et al., 2023; Wang et al., 2022). Moreover, NPM1mut transcripts often persist following intensive chemotherapy treatment, contributing to higher relapse rates (Falini et al., 2020; Wang et al., 2022). While targeted therapies, such as FLT3, BCL-2, IDH1/2, and menin inhibitors, have shown activity against this AML subtype (Falini et al., 2020; Chin et al., 2023; Wang et al., 2022), most patients relapse due to disease heterogeneity. Therefore, these challenges highlight the need for novel therapeutic strategies in affected NPM1mut AML patients.

Objectives This project aimed to evaluate and identify effective drug combinations and repurpose approved or investigational drugs to target NPM1mut AML. We integrated drug sensitivity profiles and transcriptomic, genomic, and clinical data to discover novel drug combinations.

Methods Ex vivo drug sensitivity data from 215 AML patient bone marrow (BM) samples tested against 528 small molecule inhibitors (Malani et al., 2022) were analyzed with a focus on NPM1mut AML. Drug activity was assessed using drug sensitivity scores (DSS), calculated from the area under the dose-response curve (Yadav et al., 2014). Comparative analysis of drug sensitivities for genomically defined AML subtypes was done using ANOVA. Validation of single and drug combination activity was assessed by multiparametric flow cytometric analysis using a select set of AML BM samples (n=10). These samples were chosen to include representative NPM1mut cases with co-occurring mutations FLT3 and DNMT3A. All samples were collected with informed consent, and the study was conducted in accordance with the Declaration of Helsinki.

Results Out of the 215 AML samples, 52 were NPM1mut positive. Comparative analysis of DSS between NPM1mut and NPM1 wild-type (WT) samples revealed that the NPM1mut samples exhibited increased sensitivity to 12 inhibitors. Notably, NPM1mut AML samples were significantly more sensitive to pacritinib, with a -log₁₀FDR value of approximately 2.0. Pacritinib is an oral multikinase small-molecule inhibitor approved for myelofibrosis (Mascarenhas, 2022) with potent selectivity against JAK2 and FLT3, including both FLT3 internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations (William et al., 2011). Its distinct kinase profile limits off-target effects and myelosuppression, making it an attractive candidate for FLT3-driven leukemias. Given the high prevalence of FLT3 co-mutations in NPM1mut AML, we investigated the therapeutic potential of pacritinib in this AML subtype both with and without FLT3 co-mutations.

To assess the efficacy of pacritinib across NPM1 mutation subtypes, ANOVA was performed, suggesting the drug's potential regardless of other common co-occurring mutations (p > 0.05). However, preliminary multiparametric flow cytometric results showed that pacritinib had limited efficacy in FLT3 co-mutated samples. Based on these findings, we hypothesize that differential proteomic signatures underlie variable drug responses particularly in the context of the co-mutations. Ongoing studies aim to elucidate these mechanisms.

We further tested pacritinib in combination with venetoclax ex vivo at low concentrations (pacritinib 30 or 100 nM + venetoclax 10 or 100 nM), which showed enhanced activity compared to either drug alone in NPM1mut AML. Notably, the combination was generally effective regardless of individual sample sensitivity or resistance to either drug alone.Conclusion Pacritinib, approved for myelofibrosis and investigated in FLT3 mutated AML (Jeon et al., 2020), demonstrated enhanced activity in NPM1mut AML in our study. Our preliminary data suggest that combining pacritinib with venetoclax may improve therapeutic efficacy, supporting further exploration of this combination as a targeted strategy for NPM1mut AML. Given that genomic mutations alone do not fully account for differential drug responses, ongoing proteomic analyses aim to uncover mechanisms of sensitivity and resistance, which may guide future precision therapies.