LAUSR

Acute myeloid leukemia (AML) is a life-threatening disease with limited treatment opportunities. FMS-like tyrosine kinase 3 with internal tandem duplications (FLT3ITD) is a constitutively active receptor tyrosine kinase, found in 25–30% of AML cases and promoting leukemia. Little is known about the status and role of protein-tyrosine phosphatases (PTP), the potential counter players of FLT3ITD in this disease. An abundantly expressed PTP in AML cells is SHP1/PTPN6 [1]. Physiological functions of SHP1 comprise regulation of the signaling of multiple cytokine and other cell surface receptors. Mice with genetically impaired SHP1 activity suffer from inflammation, autoimmunity, and leukemia (reviewed in reference [2]). We have assessed the role of SHP1/PTPN6 in AML cell lines harboring FLT3ITD or wild-type FLT3. As shown in Supplementary Fig. S1A (Online Supplementary Information), SHP1 depletion in cells harboring wild-type FLT3 led to mildly enhanced FLT3-ligand or cytokine (IL-3) stimulated growth. In contrast, SHP1 depletion in FLT3ITDexpressing cells did not augment but rather tended to attenuate growth (Supplementary Fig. S1A). Similar observations were made in colony assays, where SHP1 depletion had mild attenuating effects in FLT3ITDexpressing 32D cells (Supplementary Fig. S1B), and a significant inhibitory effect in MOLM13 cells (Supplementary Fig. S1C). We then analyzed the effect of shRNAmediated SHP1 depletion in the syngeneic 32D-C3H/HeJ model of myeloproliferative disease using FLT3ITDtransduced 32D cells, which also express GFP. Injected mice were either sacrificed 10 days p.i. and bone marrow and spleen were analyzed for GFP-positive cells (Fig. 1a) or animal survival was monitored (Fig. 1b). SHP1-depleted cells were greatly compromised in generating a myeloproliferative disease. The amounts of GFP-positive SHP1depleted cells were strongly reduced in bone marrow and spleen, and survival of mice was significantly prolonged (Fig. 1a, b, respectively). We further employed a bona fide model of leukemia [3], which is based on injection of MLLAF9/FLT3 leukemic stem cells into sublethally irradiated secondary recipient mice. Consistently, SHP1 depletion with shRNA delayed disease development, as revealed by reduced white blood-cell count (Fig. 1c) and improved survival (Fig. 1d). Taken together, SHP1 appears to be required for the development of an FLT3ITD-driven myeloproliferative disease and acute leukemia in two different disease models in vivo. We proceeded to identify underlying mechanisms for this phenotype. SHP1-depleted cells homed normally to the bone marrow (Supplementary Fig. S2A). Interestingly, spontaneous adhesion of SHP1-depleted cells to the culture dishes was observed upon generation of SHP1deficient cell pools (Supplementary Fig. S2B). We considered that this phenotype was related to the effects of These authors contributed equally: Daniela Reich, Ashok Kumar Jayavelu