Periodic Reporting for period 4 - AltCheM (In vivo functional screens to decipher mechanisms of stochastically- and mutationally-induced chemoresistance in Acute Myeloid Leukemia)
Periodo di rendicontazione: 2022-09-01 al 2024-02-29
Acute myeloid leukemia (AML), the most prevalent type of leukemia in adults, encompasses a diverse set of cancers characterized by the proliferation of myeloblasts—cells derived from hematopoietic stem cells and myeloid progenitors—constituting over 20% of cells in the blood or bone marrow. AML is notoriously resistant to front-line therapies, making it a significant challenge in hematology. The primary goal of our research project was to elucidate the poorly understood biological mechanisms that enable AML cells to resist chemotherapy and targeted therapies. By employing large-scale screening, functional genomics, and proteomics in pre-clinical models, including syngeneic and patient-derived xenograft (PDX) mouse models, we aimed to map the epigenetic, metabolic, and post-transcriptional changes, as well as the alterations in kinase kinetics and genetics, associated with therapy resistance. To further this goal, we developed an isogenic mouse model resistant to the standard therapies of anthracycline and cytarabine. Utilizing multi-omics profiling and functional genomic approaches, we identified critical gene drivers of chemoresistance. Our findings, validated in both syngeneic and humanized pre-clinical models and two cohorts of primary patient samples, highlight potential therapeutic targets to prevent and overcome relapse post-chemotherapy.
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.
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.
Overall, these accomplishments highlight the execution of cutting-edge research in our laboratory, employing advanced techniques in the field of hematology. By applying sophisticated methodologies and conducting comprehensive analyses, our research has yielded invaluable insights into the multifaceted mechanisms driving resistance to chemotherapy and targeted therapies in AML. Firstly, we identified critical epigenetic and post-transcriptional mechanisms involved in therapy resistance, elucidating how cancer cells adapt to evade treatment. Secondly, our studies extensively characterized the complex interactions between genetic alterations, epigenetic modifications, and metabolic reprogramming that contribute to resistance mechanisms. Thirdly, we explored novel intervention strategies based on these insights, informing the development of innovative therapeutic approaches to prevent AML relapse. Ultimately, our research activities have achieved national and international recognition over the past five years, allowing us to innovate and disseminate our findings to other laboratories and students in our network.