Unveiling FLT3 in B-ALL: Molecular drivers and targeted treatment opportunities

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Abstract Backgroud : The prognosis of B-cell Acute Lymphoblastic Leukemia (B-ALL) patients relapsing after Hematopoietic Stem Cell Transplant and of high-risk subtypes is dismal, and novel therapeutic options are urgently needed. In Acute Myeloid Leukemia (AML), FLT3 inhibitors (FLT3i) have shown efficacy in FLT3-mutated (mut) cases, with encouraging data also emerging in FLT3-ITD-wildtype (wt) AML with a FLT3-like transcriptional profile (Mosquera; Blood 142(2023)974–76). The potential of FLT3i in both FLT3-mut and wt B-ALL has been insufficiently explored. FLT3 is emerging as a key oncogenic driver in acute leukemias, with ALL-specific deregulation patterns supporting the need for tailored therapeutic strategies. Aim: To investigate FLT3 as a possible therapeutic target in B-ALL. Methods: We performed capture-based RNA-seq panel (Pan-Cancer, Illumina; 1385 genes) on 215 adult B-ALL samples [51 Ph-positive or carrying t(4;11)/t(1;19) and 148 Triple-Negative (TN) cases, lacking all three translocations], along with 15 healthy donors. To validate FLT3 mutations, 14 available samples underwent additional sequencing using the Extended Myeloid Solution panel (SOPHiA; 98 genes), which provides full FLT3 coverage. Drug response data for 43 pediatric B/T-ALL cell lines to 9 FLT3 inhibitors were analysed from the FORALL database (Karolinska Institutet; covering 13 B-ALL and 5 T-ALL subtypes). In vitro assays with increasing concentrations of 6 FLT3i for 24, 48, 72h [Gilteritinib (Gil), Midostaurin, Crenolanib, Sorafenib, Quizartinib, Ponatinib (Pon)] and Venetoclax (Ven) were performed on primary patient samples (n=29; FLT3-mut n=5/29), 11 B-ALL wt cell lines, 2 B-ALL mut (NALM6-mut, KASUMI-10), and 4 AML cell lines (OCI-AML3 wt; MV-4-11, MOLM-13, MONO-MAC6 FLT3-mut). Results: Our RNA-seq variant analysis revealed that, after TP53 and KMT2A, FLT3 is the 3rd most mutated gene. We found 15 FLT3 mut in 15/148 TN B-ALL and in 1/51 Ph+ cases, distributed mainly in JM and TK domains. Overall, 10.1%of TN were mut (Ph+ 2%). 68.8% of FLT3mut pts, mainly TKD (50%; ITD 20%), potentially druggable with FLT3i. FLT3 expr. was increased in 13/16 mut cases compared to donors and wt pts, with higher expression in ZNF384, KMT2Ar, CEBPEr B-ALL. More than a third of mut cases presented an IKZF1-plus profile, half of the cases had alterations in the cohesin and CTCF genes which are part of the TAD boundary «machinery» and most of the cases had alterations in epigenetic genes. Analysis of the FORALL dataset reveals diverse FLT3i responses across pediatric ALL cell lines. Ponatinib, midostaurin, and quizartinib were the most effective, especially inKMT2A-r and some T-ALL lines. KASUMI-10, the only FLT3-mut B-ALL model, was highly sensitive to all FLT3i. Other inhibitors showed variable, subtype-specific activity. Nine cell lines (7 B-ALL, 2 T-ALL) were resistant to all FLT3i, including KASUMI-2 (TCF3-PBX1) and two IGH-MYC c-lines (MN-60, TANOUE). From our results, the response to the various FLT3i and Ven is confirmed to be heterogeneous in the different 17 cell lines. REH and JIH-5 Ph negative cell lines, were highly sensitive to Pon. Interestingly, NALM-6 cells engineered with FLT3-ITD showed increased sensitivity to all FLT3i, similarly to the FLT3-mut KASUMI-10 cell line. Interestingly MHH-CALL4 wt (a Ph-like cell line model) were more sensitive to Gil compared to OCI AML3 wt, and showed the same effect compared to MV-4-11 AML mut. FLT3i testing on 29 primary B-ALL cells revealed that midostaurin, sorafenib, and quizartinib were more effective in FLT3-mut samples. All inhibitors also showed activity in FLT3-wt cases, although with generally higher IC50 values compared to mutated ones. Interestingly, gil demonstrated comparable IC50 values in both FLT3-mut and wt samples. Given the observed effect of gil as a single agent in Ph-like cell lines/primary cells, and of ponatinib (already approved in ALL), we combined them with Ven in B-ALL cell lines (representative of Ph- adult molecular subgroups) and 10 adult B-ALL primary cells at increasing concentrations. Interestingly we noticed a strongsynergistic or additive effect in tested cell lines and primary cells (n=1 mut). Conclusions: FLT3 alterations define a novel Ph-negative B-ALL subgroup with therapeutic relevance. Given the responses in FLT3-wt in addition to the mutated models, FLT3i may also benefit patients without FLT3 muts. Thanks to: Ricerca Corrente by the Italian MoH L3P1946 and AILTreviso.

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