CO08 | Flt3 as a new therapeutic target in preclinical models of pediatric BCP-ALL with PAX5 rearrangement

Abstract

Introduction: B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) is the most common pediatric cancer, with survival rates exceeding 85%. However, prognosis remains poor in refractory or relapsed cases. PAX5, a transcription factor essential for B-cell development, is frequently altered in BCP-ALL (PAX5-alt), including rearrangements (PAX5r). These lead to repression of genes typically activated by wild-type PAX5 and the upregulation of genes normally repressed by PAX5. Fms-like tyrosine kinase 3 (FLT3) gene was previously reported among PAX5-repressed genes. We aim to investigate FLT3 expression in pediatric PAX5r BCP-ALL and design an effective molecularly targeted treatment in this molecular ALL subgroup.
Methods: RNA-seq was used to identify PAX5 fusion genes. FLT3 expression was assessed in pediatric BCP-ALL patients and PDX samples derived from PAX5r cases (AIEOP-BFM ALL protocols 2000, 2009, 2017) by RNA-seq and confirmed with RT-qPCR. HTP screening platform strategy has been applied to test FLT3 inhibitors. Among them, gilteritinib was selected and tested in monotherapy and combined with chemotherapy on NALL-1 cells (PAX5::ETV6) and n=4 PAX5r samples using Annexin V/7-AAD FACS staining. Phosphoflow analysis was performed to evaluate FLT3 activation in NALL-1 cells.
Results: FLT3 expression was profiled in 599 consecutive pediatric BCP-ALL patients at diagnosis enrolled in the AIEOP-BFM ALL 2017 study. PAX5r patients (n=26) showed significantly higher median FLT3 expression compared to the whole cohort of patients, and levels comparable to KMT2Ar, ZNF384r, and high-hyperdiploid ALL cases, all known to have high FLT3 expression profiles. These results were confirmed by RT-qPCR in primary PAX5r samples and PAX5r PDXs. None of 10 PAX5r with the highest FLT3 expression levels resulted positive for the most common FLT3-ITD mutation by RT-PCR.
16 PAX5r PDXs were used to perform a high-throughput screening of six FLT3 inhibitors to assess drug sensitivity. Among these, gilteritinib, demonstrated a promising efficacy and toxicity profile. In vitro experiments with NALL-1 cells and PAX5r PDX blasts (n=4) revealed gilteritinib's potent cytotoxicity at nanomolar concentrations, both as a single agent and in combination with chemotherapy (dexamethasone and asparaginase). Phosphoflow analysis further confirmed the high FLT3 expression and constitutive basal activation as measured by phospho-FLT3 levels in NALL-1 cells. Following gilteritinib treatment, a significant reduction in FLT3 phosphorylation was observed (-57.5%, p<0.05).
Conclusions: FLT3 is highly expressed in PAX5r BCP-ALL, as already known in KMT2Ar and ZNF384r ALL cases. Ex vivo treatment with gilteritinib induced apoptosis in leukemic cells, both as single-agent and in combination with chemotherapeutics, demonstrating synergistic (dexamethasone) or additive (asparaginase) effects. Further in vivo studies are needed to confirm its efficacy in this molecular ALL subgroup.