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Supplementary Data from Phosphorylation of SHP2 at Tyr62 Enables Acquired Resistance to SHP2 Allosteric Inhibitors in FLT3-ITD–Driven AML

The protein tyrosine phosphatase SHP2 is crucial for oncogenic transformation of acute myeloid leukemia (AML) cells expressing mutated receptor tyrosine kinases. SHP2 is required for full RAS-ERK activation to promote cell proliferation and survival programs. Allosteric SHP2 inhibitors act by stabilizing SHP2 in its autoinhibited conformation and are currently being tested in clinical trials for tumors with overactivation of the RAS/ERK pathway, alone and in various drug combinations. In this study, we established cells with acquired resistance to the allosteric SHP2 inhibitor SHP099 from two FLT3-ITD (internal tandem duplication)-positive AML cell lines. Label-free and isobaric labeling quantitative mass spectrometry–based phosphoproteomics of these resistant models demonstrated that AML cells can restore phosphorylated ERK (pERK) in the presence of SHP099, thus developing adaptive resistance. Mechanistically, SHP2 inhibition induced tyrosine phosphorylation and feedback-driven activation of the FLT3 receptor, which in turn phosphorylated SHP2 on tyrosine 62. This phosphorylation stabilized SHP2 in its open conformation, preventing SHP099 binding and conferring resistance. Combinatorial inhibition of SHP2 and MEK or FLT3 prevented pERK rebound and resistant cell growth. The same mechanism was observed in a FLT3-mutated B-cell acute lymphoblastic leukemia cell line and in the inv(16)/KitD816Y AML mouse model, but allosteric inhibition of Shp2 did not impair the clonogenic ability of normal bone marrow progenitors. Together, these results support the future use of SHP2 inhibitor combinations for clinical applications.Significance:These findings suggest that combined inhibition of SHP2 and FLT3 effectively treat FLT3-ITD–positive AML, highlighting the need for development of more potent SHP2 inhibitors and combination therapies for clinical applications.

Supplementary Table from Phosphorylation of SHP2 at Tyr62 Enables Acquired Resistance to SHP2 Allosteric Inhibitors in FLT3-ITD–Driven AML

Supplementary Figure from Phosphorylation of SHP2 at Tyr62 Enables Acquired Resistance to SHP2 Allosteric Inhibitors in FLT3-ITD–Driven AML

SHP2 is a cytoplasmic non-receptor tyrosine phosphatase involved in the propagation of extracellular signaling through receptor tyrosine kinases. Aberrant SHP2 activity has been identified as a driver in multiple cancers and SHP2 has also been implicated in the PD-1/PD-L1-mediated exhaustion of effector T-cells, leading to immune system evasion of tumors. Recently, we reported the identification of SHP099, an allosteric inhibitor of SHP2 with in in vivo efficacy against multiple RTK-driven tumor xenograft models. Here we report the use of alternate screening paradigms to identify a novel allosteric inhibitor which binds to a previously uncharacterized pocket on SHP2. Like SHP099, the second allosteric inhibitor stabilizes a closed conformation of SHP2, which blocks access to the phosphatase active site. Structure based drug design led to improvements in potency, and combination studies in biochemical, biophysical and cellular assays confirm dual occupation of SHP099 and the second allosteric molecule, resulting in improved potency. This work highlights a rare opportunity for dual occupation of inhibitors for a single target and provides additional tools for the exploration of SHP2 biology. Citation Format: Michelle Fodor, Edmund Price, Ping Wang, Hengyu Lu, Andreea Argintaru, Zhouliang Chen, Meir Glick, Huai-Xiang Hao, Mitsunori Kato, Robert Koenig, Jonathan R. LaRochelle, Gang Liu, Eric McNeill, Dyuti Majumdar, Gisele Nishiguchi, Lawrence Perez, Greg Paris, Christopher Quinn, Timothy Ramsey, Martin Sendzik, Michael Shultz, Sarah Williams, Travis Stams, Stephen C. Blacklow, Matthew J. LaMarche, Michael G. Acker. Simultaneous inhibition of SHP2 phosphatase at two allosteric sites [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2808.

The non-receptor protein tyrosine phosphatase (PTP) SHP2 is an important component of RTK signaling in response to growth factor stimulus and sits just upstream of the RAS-MAPK signaling cascade. The first oncogenic phosphatase to be identified, SHP2 is dysregulated in multiple human diseases including the developmental disorders Noonan and Leopard syndromes, as well as leukemia, lung cancer and neuroblastoma where aberrant activity of SHP2 leads to uncontrolled MAPK signaling. Cancer-associated activating mutations in SHP2 impart an “auto-on” state of the enzyme, boosting basal activity by shifting the equilibrium away from the auto-inhibited state. Reduction of SHP2 activity through genetic knockdown suppresses tumor growth, validating SHP2 as a target for cancer therapy. SHP099, a recently reported potent and selective allosteric inhibitor of SHP2, stabilizes the auto-inhibited form of SHP2 through interactions with the N-terminal SH2 and C-terminal PTP domains of the protein. SHP099 suppresses MAPK signaling in RTK amplified cancers resulting in suppressed proliferation in vitro and inhibition of tumor growth in mouse tumor xenograft models. Together, these data demonstrate the therapeutic potential of SHP2 inhibition in the treatment of cancer and other RAS/MAPK-linked diseases. Citation Format: Michael G. Acker, Ying-Nan P. Chen, Matthew J. LaMarche, Ho Man Chan, Peter Fekkes, Jorge Garcia-Fortanet, Jonathan R. LaRochelle, Brandon Antonakos, Christine Hiu-Tung Chen, Zhuoliang Chen, Vesselina G. Cooke, Jason R. Dobson, Zhan Deng, Fei Feng, Brant Firestone, Michelle Fodor, Cary Fridrich, Hui Gao, Huai-Xiang Hao, Jaison Jacob, Samuel Ho, Kathy Hsiao, Zhao B. Kang, Rajesh Karki, Mitsunori Kato, Jay Larrow, Laura R. La Bonte, Gang Liu, Shumei Liu, Dyuti Majumdar, Matthew J. Meyer, Mark Palermo, Minying Pu, Edmund Price, Subarna Shakya, Michael D. Shultz, Kavitha Venkatesan, Ping Wang, Markus Warmuth, Sarah Williams, Guizhi Yang, Jing Yuan, Ji-Hu Zhang, Ping Zhu, Stephen C. Blacklow, Timothy Ramsey, Nicholas J. Keen, William R. Sellers, Travis Stams, Pascal D. Fortin. Conformational activation and allosteric inhibition of SHP2 in RTK-driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2084. doi:10.1158/1538-7445.AM2017-2084