TNO155 is a selective SHP2 inhibitor to target PTPN11-dependent oral squamous cell carcinoma

Abstract

Oral squamous cell carcinoma (OSCC) is known to be driven by multiple intricated receptor tyrosine kinases (RTKs) including EGFR, PI3K/AKT and MAPK signaling pathways. However, whilst targeting EGFR with cetuximab has been approved for the treatment of OSCC, other single-agent inhibitors of the RTKs have shown modest effects in improving survival. From the genome-wide CRISPR/Cas9 screen on 21 OSCC cell lines, we have identified PTPN11 among the top essential genes in OSCC. PTPN11 encodes for SHP2, a phosphatase that acts as a master signal transducer, downstream of various RTKs. Although PTPN11 overexpression has been reported in OSCC, little is known about its role as an essential gene for OSCC survival and its potential as a therapeutic target. Herein, we confirmed that PTPN11 is an essential gene in OSCC where its deletion significantly impacted cell survival. We evaluated three SHP2 inhibitors on 21 OSCC cell lines and found TNO155 to be significantly associated with CRISPR dependency score. We showed that TNO155 caused dose-dependent suppression on p-ERK and p-MEK, and suppresses the JAK/STAT pathway via downregulating p-JAK1, p-STAT1, p-STAT3. Furthermore, we confirmed that the combination of the mTOR inhibitor, everolimus with TNO155 is synergistic in OSCC. In summary, PTPN11 is a promising therapeutic target in OSCC that can be selectively targeted by SHP2 inhibitor such as TNO155. Our findings on the use of mTOR inhibitor, everolimus to overcome resistance to TNO155 are essential to inform on next phases of clinical trials which is warranted for the treatment of OSCC.

Keywords: Allosteric inhibitor; Drug resistance; Essential genes; OSCC; PTPN11; Protein tyrosine phosphatase; RTK signaling; SHP2.

Fig. 1

Confirmation of PTPN11 as an essential gene for OSCC survival. (A) Plot of number of dependent OSCC cell lines based on our previous CRISPR/Cas9 screen of 21 OSCC lines (Chai et al., 2020), for the essential genes that are enriched in the lineage of upper aerodigestive tract (DepMap Public 22Q4, https://depmap.org/portal/). (B) Scatter plots of CRISPR scores of PTPN11 and other genes that are targets of drugs being tested for OSCC. (C) Co-competition assays between non-transduced cells with either Non-targeting (NT) or PTPN11 sgRNAs-transduced OSCC to validate dependency on PTPN11. The percentage of BFP-positive transduced cells obtained at different time points were normalized to the day 4 or 6 readings for respective sgRNAs. (D) Western blots showing depletion of the SHP2 level, the protein encoded by PTPN11 gene upon sgRNA-mediated knockout. Actin is used as loading control. NT – Non-targeting sgRNAs; “P1k” – PTPN11_sg1k”; “P2b” – PTPN11_sg2b. (E) Cell viability assay of OSCC cells upon PTPN11 knockout with P1k and P2b. (F) Colony forming assay of OSCC cells upon PTPN11 knockout with P1k and P2b. (G) Apoptosis assay of OSCC cells upon PTPN11 knockout with P1k and P2b.

Fig. 2

TNO155, an inhibitor of SHP2 exhibit selective sensitivity in OSCC. (A) IC50 of three SHP2 inhibitors (SHP099, TNO155 and RMC4550), were determined for 21 OSCC cell lines using MTT assay. Data shown is mean ± standard deviation (in μM). (B) Correlation plot of PTPN11 CRISPR scores against log₁₀ (lg) IC50 of SHP099/TNO155/RMC4550 IC50 (μM). (C) Dose-dependent effects of TNO155 on two selective “sensitive” lines – ORL-195 and SCC-9; and two “resistant” lines – BICR10 and PE/CA-PJ15 were measured by apoptosis assay. Bar charts show the percentage of apoptotic cells at increasing concentration of TNO155. Error bars are mean±SD of two biological repeats with technical triplicates. Unpaired student's t-test was used with untreated control (0 μM TNO155) as comparator. ∗p: <0.05, ∗∗: p < 0.01, ∗∗∗: not significant (ns). (D) Dose-dependent effect of TNO155 on colony formation assay in sensitive and resistant OSCC cell lines.

Fig. 3

Effect of TNO155 on phosphorylation levels of kinases in its downstream and parallel signaling pathways. (A) Western blot shows the effect of various TNO155 concentration on MEK and ERK phosphorylation levels upon 1-, 6- and 24-h treatment, in sensitive and resistant OSCC lines. (B) Phosphorylation arrays of DMSO-treated (control) and TNO155-treated OSCC lines reveal the overall effects of TNO155 on the downstream (MAPK) and other related phosphorylation signaling pathways (AKT, JAK/STAT, and TGFβ) and identify potential markers that are distinctive between sensitive and resistant lines. (C) Graphical representation of the quantified changes on the phosphorylated markers upon TNO155 treatment in the sensitive and resistant lines. Icons in the boxes represent whether the markers expression was upregulated (green upward arrow), downregulated (red downward arrow), or unchanged (yellow rightward arrow) upon TNO155 treatment. Differences in baseline expression were also indicated by the font colours of the markers, by comparing the signal intensities between the average of sensitive lines and average of resistant lines. Red font indicate that the markers have significantly higher level among sensitive lines than resistant lines; while green font indicate that the markers have significantly higher level among resistant lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Combination of TNO155 and everolimus are synergistic in OSCC lines. (A) Matrix of cell viability across various combinations of TNO155 (SHP2 inhibitor) and everolimus (mTOR inhibitor) in BICR10 and PE/CA-PJ15, suggesting a synergistic effect. (B) Plot of combination index (CI) against fraction affected (Fa) of TNO155 (0.02 μM, 0.1 μM, 0.48 μM, 2.4 μM and 12 μM) and everolimus (0.0003 μM and 0.006 μM) combinations in BICR10 and PE/CA-PJ15. Only doses that are lower than single-agent IC50 is plotted here. (C) Colony formation assays at various combinations of TNO155 and everolimus showing the synergistic effect of the combinations. (D) Western blot showing the effect of the TNO155 and everolimus combinations on the phosphorylation levels of key downstream markers.