Acute myeloid leukemia remains a lethal disease, especially for patients who do not respond to intensive chemotherapy. Despite significant new data resulting from next-generation DNA sequencing techniques, it remains difficult to link specific genetic mutations to the molecular mechanisms that drive aberrant proliferation of leukemic cells. Moreover, some 50% of AML patients who display a normal karyotype and who lack mutations in key genes such as FLT3 or NPM1 are resistant to induction chemotherapy. In contrast to gene sequencing, phosphoproteomic approaches provide a direct and quantitative measure of the signaling molecules that mediate leukemogenesis, circumventing the need to decipher 'driver' mutations from the very large number of non-functional 'passenger' DNA lesions that accumulate in tumors. In preliminary studies for this research proposal, we discovered novel mechanisms by which signaling pathways support AML cell growth and survival. In related work, we have developed DEEP SEQ mass spectrometry, a scalable platform that provides true, genome-wide proteome quantification. Consistent with the objectives described in FOA PA-12-220, which encourages development and improvement of specific technologies for quantitative detection of novel biomarkers associated with hematopoietic malignancies, with a focus on early detection, prediction of progression, and recurrence of hematopoietic malignancies, especially in high-risk individuals, we will further improve and utilize our DEEP SEQ platform to identify phosphorylation-based, functional biomarkers in primary tumors of high-risk AML patients. We will validate our phosphoproteomic data using capillary nanoimmunoassays and targeted mass spectrometry, analytical formats suitable for point-of-care clinical applications. Our novel discovery and validation strategy will capture the signaling pathways associated with therapy failure in high-risk AML patients, and lay the foundation for translation of our phosphoproteomic data into improved diagnostics. Our study plan has two specific aims:
Specific Aim 1 is to establish phosphoproteomic signatures of chemoresistance and therapy failure in diagnostic specimens from patients with AML. We will use DEEP SEQ mass spectrometry to identify phosphorylation markers associated with chemoresistance and therapy failure. Importantly, we have secured collaborative agreements with colleagues at four major cancer centers to access primary AML patient samples that are well-annotated with respect to genetic background and clinical outcomes.
Specific Aim 2 is to validate phosphoproteomic markers and access their prognostic performance in independent cohorts of AML patients. We will validate our phosphoproteomic biomarkers across a larger, independent patient cohort to determine prognostic performance. To facilitate eventual translation to clinical application we will perform these validation studies using the complementary techniques of capillary nanoimmunoassays and targeted mass spectrometry.
We will further develop and utilize DEEP SEQ mass spectrometry to identify phosphorylation-based signatures of chemo-resistance in a subset of high-risk acute myeloid leukemia patients. These data will provide a direct and quantitative measure of the signaling molecules that mediate leukemogenesis, circumventing the need to decipher 'driver' mutations from the very large number of non-functional 'passenger' DNA lesions that accumulate in tumors. In the fullness of time these data will improve patient diagnosis and stratification, inform drug efficacy studies, and aid in the search for early detection AML biomarkers.
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