P395: pharmaceutical targeting of ras in acute myeloid leukaemia

Background: Acute Myeloid Leukaemia is characterised by a small number of co-occurring driver mutations which combine to make a diverse collection of disease subtypes. Mutations in genes associated with cell signalling are a key group of driver mutations and lead to the constitutive activation of these proteins and their associated signalling cascades. RAS mutations occur in 6% of AML and confer a proliferative advantage. However, 36% of AMLs include mutations which lead to activated RAS such as FLT3 mutations and are associated with a worse outcome after induction chemotherapy1. Inhibitors which bind to mutated and activated members of the RAS protein family to block the interaction between the protein and its downstream targets have been developed2 which allow us to investigate the effects of pan-RAS inhibition in Acute Myeloid Leukaemia. These Pan-RAS inhibitors inhibit RAS-RAF and RAS-PI3K signalling leading to reduced phosphorylation of ERK and AKT. Aims: 1. We have used novel specific and panRAS RAS inhibitors designed in the Rabbitts lab followed by system-wide analyses to identify the specific gene regulatory network of mutant RAS AML and identify RAS-responsive genes 2. We have interrorgated key transcription factor nodes with RNAi Methods: Primary cells were co-cultured on primary hMSCs for perturbation experiments. Fresh blood or bone marrow samples from patients with AML were purified and sorted for CD34 or CD117, these samples were profiled by RNA-seq an ATAC-seq. Data from multiple samples with RAS mutations was integrated and compared to data from healthy CD34+ PBSCs to establish the gene regulagtory network. Results: In vitro panRAS inhibitors cause cytotoxicity in primary AML samples with RAS activating mutations whilst cells with WT RAS exhibit reduced sensitivity. An exception to this finding are cells with FLT3-ITD mutations, which were also sensitive to RAS inhibition. This is of particular interest as patients treated with FLT3 inhibitors can relapse with a RAS mutant subclone. To understand the role of RAS mutations in AML we have constructed a gene regulatory network from the genomic and transcriptomic profiling of primary cell samples from patients which has allowed us to identify a core network of transcription factors. The direct association of aberrant RAS signalling with these transcription factors has been determined through perturbation experiments using RAS inhibitors, and by targeting key transcription factor nodes with RNAi. These include transcription factors ETV5 and KANK1, both of which have been linked to RAS signalling in solid tumour models3,4. Summary/Conclusion: These experiments will identify targets of the RAS pathway essential for the survival of AML cells. 1. Ball, B.J., et al. (2019), “RAS Mutations Are Independently Associated with Decreased Overall Survival and Event-Free Survival in Patients with AML Receiving Induction Chemotherapy.”Blood, 134(Supplement_1): 18-18. 2. Cruz-Migoni, A., et al. (2019), “Structure-based development of new RAS-effector inhibitors from a combination of active and inactive RAS-binding compounds.” Proceedings of the National Academy of Sciences116(7): 2545-2550. 3. Mus, L.M., et al. (2020), “The ETS transcription factor ETV5 is a target of activated ALK in neuroblastoma contributing to increased tumour aggressiveness.” Sci Rep10(218) 4. Jonathan R. Dry, et al. (2010), “Transcriptional Pathway Signatures Predict MEK Addiction and Response to Selumetinib (AZD6244)”. Cancer Res; 70(6): 2264–2273