Abstract 2944: Targeting negative feedback signaling in tyrosine kinase-driven malignancies
Authors: Shojaee Seyedmehdi , Buchner Maike , Swaminathan Srividya , Chan Linda , Bothe Marle , Geng Huimin , Melnick Ari , Molkentin Jeffery , Martin Gail , Koeffler Phillip , Muschen Markus 
Published: August 2012

Abstract
Background: Negative feedback signaling molecules have been shown to limit proliferation. For instance, DUSP6 and SPRY2 attenuate signaling from surface receptors and thereby function as tumor suppressors. Here we studied mechanisms of negative feedback signaling in tyrosine kinase-driven (TKD) malignancies. Results: Studying gene expression changes by microarray analysis, RT-PCR and Western blot, we found that the DUSP6 and SPRY2 negative feedback regulator molecules are highly expressed in a wide array of TKD malignancies including solid tumors, myeloid and B cell lineage leukemia. In contrast to TKD leukemia, Non-TKD tumors lack expression of these genes and DUSP6 and SPRY2 promoters are hypermethylated in Non-TKD tumors. To study the function of DUSP6 and SPRY2 in a genetic mouse model of TKD-leukemia, we transduced bone marrow pre-B cells from DUSP6-/-, SPRY1/2-fl/fl mice and respective wildtype controls with retroviral BCR-ABL1. Defects in one of these negative feedback mediators caused profound signaling imbalances in TKD leukemia cells. For instance, Dusp6-deficient TKD-leukemia cells rapidly underwent cellular senescence. Lack of Dusp6 and Spry2 dramatically increased cellular ROS. Owing to excessively high levels of ROS, TKD-leukemia cells lacking one of these negative feedback mediators failed to initiate colonies in semisolid agar and failed to initiate leukemia in transplant recipient mice. Inducible deletion of Spry1/2 in leukemia cells resulted in rapid cell death. To test whether negative feedback signaling molecules represent a potential target for pharmacological inhibition in the treatment of TKD-leukemia and TKD solid tumors, we tested the Dusp6 small molecule inhibitor 2-benzylidene-3-(cyclohexylamino)-1-Indanone hydrochloride (BCI). At 3 μmol/l, BCI induces massive accumulation of ROS and subsequent cell death in 5 patient-derived cases of Ph+ ALL including two cases with T315I mutation. In addition, a panel of 23 TKD solid tumors carrying EGFR, Her2, PDGFRA and RET kinase is highly sensitive to BCI To test in vivo efficacy of BCI, patient-derived Ph+ ALL cells with T315I were xenografted into NOD/SCID recipient mice. Mice were treated ten times with either vehicle or 25 mg/kg BCI (i.p.). In agreement with in vitro results, treatment with BCI resulted in significant prolongation of overall survival compared to standard TKI-treatment (BCI vs TKI Nilotinib p=0.01). Conclusion: Our studies identify negative feedback mediators including DUSP6 and Spry2 as a novel therapeutic target in TKD-leukemia and TKD solid tumors. Pharmacological inhibition of negative feedback signaling represents a fundamentally novel and powerful approach to increase oncogenic signaling beyond a tolerable threshold, thus causing excessive accumulation of ROS and subsequent cell death. Normal cells lacking oncogenic tyrosine kinase signaling are insensitive to inhibition of negative feedback signaling.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2944. doi:1538-7445.AM2012-2944