P451: gene fusions and other genomic events underlie venetoclax and hypomethylating agent resistance and provide new targets in acute myeloid leukemia.

Background: Venetoclax is a BCL-2 inhibitor that promotes apoptosis in acute myeloid leukemia (AML) cells. A plethora of mechanisms responsible for resistance to venetoclax (VEN) have been identified, including expansion or gain of clones with activation of receptor tyrosine kinase (RTK) pathway, disrupted TP53 function, selection of AML populations with monocytic differentiation, MCL1 overexpression, switch to lipid metabolism, BCL-2 and BAX mutations. The contribution of rearrangements, also in co-recurrence with genomic events, to resistance remains unexplored. Aims: To discover novel VEN resistance mechanisms, in order to identify addressable molecular pathways that may restore drug sensitivity or offer novel therapeutic opportunities. Methods: After informed consent (INTHEMA protocol, NCT04298892), we collected ex-vivo samples from 21 consecutive patients that were referred to our institution at relapse or refractoriness after VEN + hypomethylating agents treatment. We performed TruSight RNA Pan-Cancer searching for fusion genes (capture technology, 1385 genes), RNA expression and expressed mutations on each sample. Fusions were validated as needed. All the patients were also analyzed with routine diagnostic NGS panel, cytogenetics and immunophenotype. Results: We identified at least one putative mechanisms of VEN resistance in 17/21 (81%) patients (figure) including the co-occurent mutations and genomic rearrangements: 14/21 patients had RTK mutations (67%; NRAS=4, PTPN11=4, CBL=4, FLT3=3, JAK2=1, cKIT=1, EPHB6=1, FGFR=1, PDGFRb=1), 1 carried BAX mutation (p.G67R; BH3 domain), 3/21 switched to monocytic phenotype, 2 patients had marker chromosomes, and no patient had TP53 mutation. Gene fusions were detected in 8/21 patients: In particular, we discovered a cryptic rearrangement of MECOM (TBL1XR1-MECOM and ETV6_MECOM) in 2 patients, a novel BCL11B fusion (BCL11B-GSDMC), a CCND2-ETV6 BIRC6-LTBP1, the reciprocal NUP98-NSD1, the canonical CBFB-MYH11 and additional transcripts with a less defined role (PSPC1-ZMYM2; PER1-DDX5; ELL-SUGP2). Interestingly, most of the genes involved in translocations, with a potential driver role in therapy resistance (MECOM, ETV6, CCND2, BCL11B, NUP98, ELL) also showed high expression levels in patients harboring the translocation. Unsupervised clustering of RNA expression data distinguished two main groups of patients (figure). A group of 5 patients (24%), showed an “activating like” signature, with CSF3R and IKZF overexpression. The other group of patients presented with a “self-renewal like” signature. Patients carrying gene fusions clustered within the “self-renewal like” group, being enriched in a subgroup (subgroup 4, 6 patients, 29%); that was also characterized by overexpression of HOXA genes. Summary/Conclusion: By the association of a deep transcriptomic characterization to conventional diagnostics, our analysis suggested novel mechanisms of resistance to VEN based combinations and detected the established ones. RTKs mutations are widespread within resistant patients, suggesting a potential therapy options. However, mutation in this pathway are redundant and not specific, thus hampering the selection of an appropriate target. Genomic analysis was able to define appealing patterns of resistance: the “activating like” signature may help define a specific target among tyrosine kinase inhibitors, while “self-renewal like” patients may benefit from histone deacetylase inhibitors (as we previously published). HOXA genes overexpression opens a novel therapeutic options for selected patients.Keywords: Fusion, Acute myeloid leukemia, Venetoclax