Abstract
Abstract 2754
The development of more effective and less toxic therapies for acute and chronic leukemias will require the identification of the molecular abnormalities contributing to leukemogenesis and the identification of drugs that specifically block the activity of these lesions. We hypothesize that aberrantly activated tyrosine kinase signaling pathways play a critical role in the pathogenesis of a substantial proportion of leukemia cases, and our preliminary data suggest that the molecular abnormalities causing aberrant kinase activation are unique in a significant number of patients. Thus, effective therapies for leukemia will need to be determined on an individual patient basis. To address this need, we have developed a function-first, small-molecule kinase inhibitor assay that can identify therapeutic targets in tyrosine kinase signaling pathways in primary leukemia samples and provide individualized therapeutic options in a clinically relevant time frame.
Methods:
To rapidly identify drug sensitivity profiles and activated kinase pathways in individual, primary leukemia samples, we have developed a small-molecule inhibitor array which includes 90 small-molecule, cell-permeable inhibitor compounds including a core of 36 tyrosine kinase inhibitors that collectively target the majority of the tyrosine kinome. Many of the inhibitors are available for clinical use or are in clinical development. Inhibitors were placed in 96-well plates at four serial dilutions to allow IC50 calculations. Three days after adding primary leukemia cells to each well, we performed a tetrazolium based cell viability assay to evaluate the effect of each inhibitor. Because most inhibitors affect multiple kinases, we utilized automated scripts to compare target specificities of compounds that uniquely decreased primary leukemia cell viability to identify potential targets.
Results:
In preliminary proof-of-principal experiments, we tested leukemia cell lines and primary leukemia samples with known activating tyrosine kinase mutations and Ba/F3 cell lines expressing activated tyrosine kinases. As expected, all cells showed hypersensitivity to compounds with activity against the primary, mutated target. In addition, downstream targets were frequently identified. For example, MKPL-1 cells, which depend on an activating CSF1R translocation for viability, also showed sensitivity to phosphoinositol 3-kinase and NFKB inhibitors. To date, we have fully analyzed approximately 150 primary myeloid and lymphoid leukemia samples. Hierarchical clustering of IC50 data for individual patients identifies activated pathways characteristic to specific leukemia subtypes. Pathways include PI3K activation in acute lymphoblastic leukemia, SRC kinase and BTK activation in chronic lymphocytic leukemia, FLT3 and KIT activation in AML patients, and MEK kinase activation in chronic myelomonocytic leukemia. Importantly, the results show heterogeneous inhibitor sensitivity profiles and potential kinase targets for individual samples even within diagnosis groups supporting a need for individualized targeted therapies. We are currently utilizing inhibitor assay results for clinical trial development. Approximately 40% of samples show sensitivity to at least one FDA approved drug in the inhibitor panel, and we are developing phase II proof-of-concept trials to test the ability of the inhibitor assay to predict effective targeted therapies for individual patients.
Conclusions:
Our data demonstrate that the small-molecule inhibitor functional assay can rapidly identify genes contributing to leukemogenesis, provide insights into their mechanism of action, and suggest therapeutic options. The unique patterns of inhibitor sensitivity in many samples support the hypothesis that tyrosine kinases and related pathways contributing to leukemogenesis in each patient may be different. These findings, in turn, support the concept that targeted therapy will be most effective when administered on an individualized basis. By utilizing our pre-clinical assay to select individualized leukemia therapies, we hope to create a platform upon which we can rapidly test the effectiveness of individualized kinase therapy and apply this information to enhance development of new drugs and new drug combinations in leukemia patients.
Disclosures:
Kovacsovics: Celator Pharmaceuticals: Research Funding. Druker:Molecular MD: Consultancy, Equity Ownership. Loriaux:Celator Pharmaceuticals: Research Funding.
Fragment-Based Discovery of a Small Molecule Inhibitor of Bruton’s Tyrosine Kinase
