To achieve the fundamental goal of our project, we developed an isogenic mouse model of AML that is resistant to the combined front-line therapies of anthracycline and cytarabine. We employed a combination of multi-omics profiling and functional genomic approaches to explore the molecular changes that occur during chemoresistance. This comprehensive analysis allowed us to identify key gene drivers of chemoresistance in AML. We demonstrated the benefits of targeting these genes to prevent and circumvent relapse in both syngeneic and humanized pre-clinical models, as well as in two cohorts of primary patient samples with AML. Our key findings include:
RNA Sequencing Insights: Initial RNA sequencing of our mouse model revealed significant changes in splicing and mRNA processing, with a distinct upregulation of 326 genes associated with therapy resistance. This finding was corroborated by analyzing relapsed AML patients from the largest public cohort available, confirming the relevance of these gene changes.
Splicing Variations and Pathways: This led us to thoroughly annotate the altered splicing events and associated biological pathways involved in chemoresistance.
Functional Screening and SRRM1 Identification: A functional screening using a pooled doxycycline-inducible shRNA library targeting our gene signature, alongside motif discovery analysis of dysregulated transcripts in chemoresistant cells, identified SRRM1 as a key factor. SRRM1, interacting with crucial alternative splicing regulators, emerged as a dependency specific to chemotherapy resistance, validated in both murine and human AML models.
Phospho-Proteomic Approach: An orthogonal phospho-proteomic approach revealed that hyperactivated PAK1 and CLK kinases regulate SRRM1 through phosphorylation at specific residues (T581, S583, and S605) in its arginine-rich domain. Consequently, chemoresistant cells showed increased sensitivity to PAK and CLK inhibitors, especially when combined with chemotherapy.
Genetic Findings and PAK1 Variant: Whole-exome sequencing uncovered that increased PAK1 activity, linked to chemoresistance, could result from a novel point variant (c.1429G>T p.(Ala477Ser)) in PAK1, enhancing its kinase activity and promoting SRRM1-mediated resistance.
Therapeutic Implications: We established that the combination of PAK1 and CLK inhibitors, mimicking SRRM1 suppression, selectively affects chemoresistant AML cells' survival and progression. This was demonstrated in both syngeneic and patient-derived xenograft mouse models, as well as in two cohorts of relapsed AML patients.
These findings provide a comprehensive understanding of the post-transcriptional changes, kinase dynamics, and genetic alterations that drive AML resistance. They suggest that combined PAK1 and CLK inhibition, either alongside or following chemotherapy, could significantly improve treatment outcomes for AML patients by disrupting SRRM1-mediated splicing adaptations.
