PS986 SYK‐DEPENDENT P‐STAT5 ACTIVITY IS REQUIRED TO MAINTAIN CLONOGENIC POTENTIAL AND OXPHOS METABOLISM IN ACUTE MYELOID LEUKEMIA (AML) CELLS
Authors: Polak A. , Bialopiotrowicz E. , Jablonska E. , Gorniak P. , Szydlowski M. , Noyszewska‐Kania M. , Piechna K. , Wozniak J. , Krzymieniewska B. , Stojak M. , Lech‐Maranda E. , Kołkowska‐Leśniak A. , Patkowska E. , Barankiewicz J. , Chlopicki S. , Juszczynski P. 
Published: June 2019

Background:

SYK kinase is an essential component of integrin b3 signaling, which maintain leukemic stem cell (LSC) transcriptional programs in AML. Genetic or pharmacologic inhibition of SYK leads to increased differentiation, reduced proliferation and apoptosis. However, the comprehensive description of mediators and effectors of SYK signaling in AML and its association with LSC maintenance is lacking.

Aims:

To characterize the role and identify key downstream mediators of SYK signaling in AML responsible for leukemia survival and LSC maintenance.

Methods:

AML cells (KG1, MOLM14, TEX) or primary AML blasts were incubated with #R406 SYK inhibitor or with vehicle for 24 h. Activity of SYK, ERK, STAT5 was assessed by western blot and/or intracellular phospho‐flow. Proliferation, apoptosis, clonogenic potential were assessed by MTS, PI staining and methylcellulose colony‐forming assay. Differentiation was assessed by Giemsa and CD14/CD15 staining, qNBT and qPCR. Constitutively active STAT5A1∗6 was electroporated into KG1. ROS level was assessed by DCF staining, mitochondrial mass was assessed by MitoTracker Green FM staining or by mitochondrial DNA content. Expression of MYC, genes involved in mitochondrial biogenesis (TFAM, NRF1, NRF2, EF‐Tu) and encoded by mitochondrial genome (ATP6, ND6) were assessed by western blot or qPCR. The oxygen consumption rate (OCR) was measured using Seahorse 96XF Analyzer.

Results:

To identify downstream mediators of SYK in AML, we assessed activity of SYK‐dependent signaling molecules in TEX, KG1 and MOLM14 cells. AML cells with active SYK kinase (pSYK) exhibited increased basal level of pERK and pSTAT5. #R406 effectively decreased phosphorylation of these proteins, reduced proliferation, clonogenic potential and induced differentiation and apoptosis. Given the role of STAT5 in self‐renewal of HSC, we assessed the contribution of STAT5 to maintenance of AML cell clonogenic potential. #R406 reduced clonogenicity of control cells, whereas in cells expressing constitutively active STAT5A1∗6, the clonogenic potential was maintained. To test whether inhibition of SYK‐STAT5 axis targets LSCs, we assessed sensitivity of LSC‐enriched TEX cells to the inhibitor and found that the compound markedly reduced their clonogenic potential and induced apoptosis. Next, we sought to identify STAT5‐dependent mechanism of LSC depletion following #R406 treatment. STAT5A1∗6‐expressing cells exhibited higher mitochondrial mass, higher expression of genes encoded by mitochondrial genome, higher expression of MYC and MYC target genes involved in mitochondrial biogenesis and, importantly, higher oxidative metabolism (OCR) as compared to control cells. LSCs sorted from TEX cells exhibited similar phenotype. In cells with forced expression of constitutively active STAT5 (STAT5A1∗6), incubation with #R406 did not significantly decrease these LSC‐associated markers, whereas mock‐transfected cells exhibited profound decrease in their abundance, indicating that STAT5 is a major effector of SYK inhibition in LSC‐like population. SYK inhibition not only reduced the expression of MYC and mitochondrial biogenesis genes in AML cells lines and primary blasts, but also reduced basal and maximal OCR in TEX, KG1 and MOLM14 cells.

Summary/Conclusion:

Taken together, we found that SYK inhibition targets LSC and reduces clonogenic potential of AML cells by decreasing STAT5 and MYC activity, and, subsequently, by decreasing mitochondrial biogenesis and oxidative metabolism.

This work was supported by NCN#2013/11/N/NZ5/03704