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Supplementary Table III: AML patient samples tested with HDM2 inhibitor, CGM097: IC50 values for CGM097.

The tumor suppressor p53 is a key regulator of apoptosis and functions upstream in the apoptotic cascade by both indirectly and directly regulating Bcl-2 family proteins. In cells expressing wild-type (WT) p53, the HDM2 protein binds to p53 and blocks its activity. Inhibition of HDM2:p53 interaction activates p53 and causes apoptosis or cell-cycle arrest. Here, we investigated the ability of the novel HDM2 inhibitor CGM097 to potently and selectively kill WT p53-expressing AML cells. The antileukemic effects of CGM097 were studied using cell-based proliferation assays (human AML cell lines, primary AML patient cells, and normal bone marrow samples), apoptosis, and cell-cycle assays, ELISA, immunoblotting, and an AML patient–derived in vivo mouse model. CGM097 potently and selectively inhibited the proliferation of human AML cell lines and the majority of primary AML cells expressing WT p53, but not mutant p53, in a target-specific manner. Several patient samples that harbored mutant p53 were comparatively unresponsive to CGM097. Synergy was observed when CGM097 was combined with FLT3 inhibition against oncogenic FLT3-expressing cells cultured both in the absence as well as the presence of cytoprotective stromal-secreted cytokines, as well as when combined with MEK inhibition in cells with activated MAPK signaling. Finally, CGM097 was effective in reducing leukemia burden in vivo. These data suggest that CGM097 is a promising treatment for AML characterized as harboring WT p53 as a single agent, as well as in combination with other therapies targeting oncogene-activated pathways that drive AML. Mol Cancer Ther; 14(10); 2249–59. ©2015 AACR.

Supplementary Table I. AML patient information.

Supplementary Figures 1-7 Supplementary Figure 1. Amplification and sequencing primers used for genotyping of p53 in AML patient samples. Supplementary Figure 2. Apoptosis data corresponding to representative bar graphs shown in Figure 1C. Supplementary Figure 3. Cell cycle analysis data corresponding to representative bar graphs shown in Figure 1D. Supplementary Figure 4. Inhibition of growth of mutant FLT3-positive AML cells by CGM097. Supplementary Figure 5. Potentiation of effects of PKC412 by CGM097 against mutant FLT3-positive AML cells. Supplementary Figure 6. Potentiation of effects of AC220 by CGM097 against mutant FLT3-positive AML cells cultured in the absence and the presence of cytoprotective SCM. Supplementary Figure 7 (A-F). Apoptosis data corresponding to representative bar graphs shown in Figure 5C. Supplementary Figure 7 (G-L). Apoptosis data corresponding to representative bar graphs shown in Figure 5C. Supplementary Figure 7 (M-R). Apoptosis data corresponding to representative bar graphs shown in Figure 5C.

Supplementary Table II: Cell lines tested with HDM2 inhibitor, CGM097: IC50 values for CGM097.

XLSX file - 243KB, This table summarizes all significant genetic features that differentiate sensitive versus insensitive cell lines for TNKSi/MEKi combination. The first sheet lists all cell lines for each group. The second sheet lists all genetic features that are enriched in the sensitive group, using a False Discovery Rate (FDR) p-value lower than 0.25.

PDF file - 68KB, Sensitivity of KRAS mutant cancer cells to TNKSi/MEKi combination. Synergistic cell lines are in red (cut-off of synergy score of 2).

PDF file - 660KB, Supplementary Figure S1. All RAS mutant cell lines have a higher sensitivity to the TNKSi/MEKi combination. Supplementary Figure S2. Validation of TNKSi/MEKi combination in KRAS mutant cancer cells. Supplementary Figure S3. Combinations with MEK inhibitor in the SW480 cell line. Supplementary Figure S4. TNKSi/MEKi combination leads to enhanced inhibition of AKT signaling activity. Supplementary Figure S5. TNKSi potentiates MEKi by releasing a feedback loop on FGFR2 signaling. Supplementary Figure S6. Consequences of combined inhibition of TNKS and MEK on FGFR2 and AKT signaling pathways in KRAS mutant cell lines.

PDF file - 123KB

XLSX file - 234KB, This table shows the features of all 138 cancer cell lines tested for the TNKSi/MEKi combination in the unbiased combination screen. Cell line name, lineage, RAS mutation status and synergy score for the TNKSi/MEKi combination are represented. Cell lines are ranked according to their synergy score.

PDF file - 81KB, List of identified synergistic cell lines for TNKSi/MEKi combination in the large-scale combination screen and their KRAS status. KRAS mutants are in red.

PDF file - 54KB, Chemical inhibitors used in the study.

The tumor suppressor p53 is a key regulator of apoptosis and functions upstream in the apoptotic cascade by both indirectly and directly regulating Bcl-2 family proteins. In cells expressing wild-type (WT) p53, the HDM2 protein binds to p53 and blocks its activity. Inhibition of HDM2:p53 interaction activates p53 and causes apoptosis or cell-cycle arrest. Here, we investigated the ability of the novel HDM2 inhibitor CGM097 to potently and selectively kill WT p53-expressing AML cells. The antileukemic effects of CGM097 were studied using cell-based proliferation assays (human AML cell lines, primary AML patient cells, and normal bone marrow samples), apoptosis, and cell-cycle assays, ELISA, immunoblotting, and an AML patient–derived in vivo mouse model. CGM097 potently and selectively inhibited the proliferation of human AML cell lines and the majority of primary AML cells expressing WT p53, but not mutant p53, in a target-specific manner. Several patient samples that harbored mutant p53 were comparatively unresponsive to CGM097. Synergy was observed when CGM097 was combined with FLT3 inhibition against oncogenic FLT3-expressing cells cultured both in the absence as well as the presence of cytoprotective stromal-secreted cytokines, as well as when combined with MEK inhibition in cells with activated MAPK signaling. Finally, CGM097 was effective in reducing leukemia burden in vivo. These data suggest that CGM097 is a promising treatment for AML characterized as harboring WT p53 as a single agent, as well as in combination with other therapies targeting oncogene-activated pathways that drive AML. Mol Cancer Ther; 14(10); 2249–59. ©2015 AACR.

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

Tankyrases (TNKS) play roles in Wnt signaling, telomere homeostasis and mitosis, and are therefore considered as attractive targets for anti-cancer treatment. Using unbiased combination screens in a large panel of cancer cell lines, we have identified a strong synergy between TNKS and MEK inhibitors in KRAS mutant cancer cells. Our study uncovers a novel function of TNKS in the relief of a feedback loop induced by MEK inhibition on FGFR2 signaling pathway. Moreover, dual inhibition of TNKS and MEK leads to more robust apoptosis and anti-tumor activity both in vitro and in vivo than effects observed by previously reported MEK inhibitor combinations. Altogether, our data provides a strong rationale for combined targeting of TNKS and MEK in KRAS mutant cancers. Citation Format: Wenlin Shao, Marie Schoumacher, Kristen Hurov, Joseph Lehar, Yan Yan-Neale, Yuji Mishina, Dmitriy Sonkin, Joshua Korn, Daisy Flemming, Michael Jones, Brandon Antonakos, Vessilina Cooke, Mark Stump, Nika Danial, William Sellers. Inhibiting TNKS sensitizes KRAS mutant cancer cells to MEK inhibitors by suppressing FGFR2 feedback signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-107. doi:10.1158/1538-7445.AM2014-LB-107