Negative Feedback Signaling Enables Leukemic Transformation by Oncogenic Tyrosine Kinases
Authors: Shojaee Seyedmehdi , Buchner Maike , Swaminathan Srividya , Geng Huimin , Chan Linda , Bothe Marle , Chen Zhengshan , Melnick Ari , Molkentin Jeffery , Martin Gail , Koeffler H. Phillip , Muschen Markus 
Published: October 2019

Abstract
Abstract 1352

Background:
Oncogenic tyrosine kinases drive malignant transformation in large subsets of hematological malignancies (e.g. BCR-ABL1, FLT3ITD) and are often associated with poor clinical outcome. Current efforts to improve therapeutic options for the treatment of tyrosine kinase-driven (TKD-) leukemia are almost entirely focused on the development of more potent tyrosine kinase inhibitors (TKI) with the goal to reduce oncogenic signaling below a minimum threshold that is required for the survival of TKD-tumor cells. Here we report the surprising finding that hyperactivation of oncogenic tyrosine kinase signaling is likewise toxic when it surpasses a previously unrecognized upper threshold of maximum tolerable signaling strength. Negative feedback signaling molecules have been shown to limit tyrosine kinase signaling strength. 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:
We identified the sprouty family inhibitor of Ras signaling Spry2, the dual-specificity phosphatase Dusp6 and the Ets transcription factor Etv5 as central components of a common negative feedback signaling module that is required to calibrate signaling strength downstream of oncogenic tyrosine kinases in ALL and CML. In genetic experiments, we demonstrated that negative feedback to oncogenic tyrosine kinase signaling is required for malignant transformation and the ability of leukemia cells to form colonies. In the absence of adequate negative feedback, hyperactive tyrosine kinase signaling leads to accumulation of ROS, activation of p53/DNA damage response, cellular senescence and massive activation of the pro-apoptotic MAP kinases JNK and p38a.
Studying gene expression changes by microarray analysis, RT-PCR and Western blot, we found that the DUSP6, SPRY2 and ETV5 negative feedback regulator molecules are highly expressed in a wide array of myeloid and B cell lineage leukemia. In contrast to TKD leukemia, Non-TKD leukemias lack expression of these genes and DUSP6 and SPRY2 promoters are hypermethylated. To study the function of Dusp6, Spry2 and Etv5 in a genetic mouse model of TKD-leukemia, we transduced bone marrow pre-B cells from Dusp6−/−, Spry2fl/fland Etv5−/−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 activation of Cre-mediated deletion of Spry2 in leukemia cells resulted in rapid cell death.

Clinical relevance:
To test whether negative feedback signaling molecules represent a potential target for pharmacological inhibition in the treatment of TKD-leukemia, we tested the Dusp6 small molecule inhibitor 2-benzylidene-3-(cyclohexylamino)-1-Indanone hydrochloride (BCI). At 3mmol/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. 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=0.008).

Conclusion:
Our studies identify negative feedback mediators including DUSP6, Spry2 and ETV5 as a novel therapeutic targets in TKD-leukemia, e.g. Ph+ ALL. 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 spared in this approach since they do not harbor an oncogenic tyrosine kinase and are not prone to excessive signaling strength.

Disclosures:
No relevant conflicts of interest to declare.