Allosteric SHP2 Inhibitor, IACS-13909, Overcomes EGFR-Dependent and EGFR-Independent Resistance Mechanisms toward Osimertinib Free

The Src homology 2 domain-containing phosphatase 2 (SHP2, encoded by PTPN11) is a critical regulator of oncogenic MAPK signaling. The SHP2 protein contains N-terminal and C-terminal Src homology 2 (SH2) domains, a protein tyrosine phosphatase (PTP) domain, and a C-terminal tail. The inactive SHP2 protein is maintained in a closed state by interdomain interactions between the N-terminal SH2 and PTP domains, preventing substrate access to the active site. SHP2 mutations at the interface of the SH2 and PTP domains, such as the somatic mutations found in juvenile myelomonocytic leukemia and the germline mutations found in Noonan Syndrome, open and activate the protein (1, 2).

In addition to being an oncogenic driver in rare malignancies, SHP2 critically mediates MAPK pathway signaling downstream of a broad spectrum of receptor tyrosine kinases (RTK), including EGFR, HER2, MET, and PDGFR (3–7). The intracellular phospho-tyrosine residues on activated RTKs interact with the SH2 domains on wild-type (WT) SHP2, resulting in an open, active conformation of SHP2, and subsequent activation of the downstream MAPK signaling cascade. SHP2 also sits upstream of RAS in the MAPK pathway and full activation of RAS requires input from SHP2, particularly the nucleotide cycling mutant of RAS (i.e., G12C; refs. 8–11).

The development of multiple generations of tyrosine kinase inhibitors (TKI) has transformed the clinical landscape of non–small cell lung cancer (NSCLC), yet acquired resistance remains as a major challenge. Osimertinib is a mutant-selective, third-generation EGFR inhibitor (EGFRi) that targets both EGFR-activating mutations (e.g., exon 19 deletion and L858R) and EGFR-dependent on-target resistance mutation toward the first-generation EGFRi (i.e., T790M; ref. 12). It is currently a first-line therapy for EGFR-mutant (EGFR^mut) NSCLC, with average progression-free survival of approximately 19 months in previously untreated patients (13). Clinical and preclinical studies have revealed numerous resistance mechanisms. Among these, EGFR-dependent mechanisms such as resistance mutations in EGFR (e.g., C797S and reversal to WT EGFR) occur in 20%–50% of relapsed patients. Other clinically observed resistance mechanisms, which also apply to earlier generations of EGFRi, include activation of alternate RTKs (e.g., MET, FGFR, HER2, and IGF1R), PIK3CA mutations, and mutations in RAS/RAF pathway that maintain downstream ERK activation (14–16). In addition, nonsignaling mechanisms such as epithelial–mesenchymal transition (EMT), acquisition of stem-like properties, and metabolic rewiring have also been reported in preclinical models (17, 18). Importantly, it has been noted that tumor from a single patient may harbor more than one resistance mechanisms (16, 19), suggesting that with oncogenic shock from EGFRi, the tumor may switch to multiple alternate drivers. For example, EGFR C797S mutation and MET amplification have been reported to coexist in the same tumor sample from a patient who relapsed on osimertinib treatment (16). The heterogeneity and plasticity in resistance mechanisms make it challenging to treat patients with a therapy targeting a single RTK.

Because SHP2 critically mediates the signaling of multiple RTKs, and several resistance mechanisms toward osimertinib are through RTK signaling, we hypothesize that a SHP2 inhibitor may be effective in addressing the heterogeneous mechanisms of osimertinib resistance. In this study, we report the discovery of IACS-13909, a novel, potent, and selective allosteric inhibitor of SHP2, that suppresses signaling through the MAPK pathway and inhibits proliferation of RTK-activated tumors in vitro and in vivo. Importantly, we provide preclinical data showing IACS-13909, either administered as a single agent or in combination with osimertinib, potently suppresses tumor cell proliferation in vitro and causes tumor regression in vivo in tumors with EGFR-dependent and EGFR-independent resistance mechanisms.

Additional/detailed methods are provided in Supplementary Data.

Phosphatase activity of full-length SHP2 or SHP2 phosphatase domain was measured using fluorogenic 6,8-difluoro-4-methylumbelliferyl phosphate (Molecular Probes) as the substrate. Detailed method is described Supplementary Data.

Cocrystals of SHP2:IACS-13909 were generated and a 2.4-Å structure was solved by X-ray crystallography. Details for crystal generation, structure determination, and data analysis are provided in Supplementary Data.

All cell lines, unless specified, were obtained from an internal cell bank, which conducted short tandem repeat (STR) finger printing and PCR-based Mycoplasma testing on all cryopreserved batches. STR finger printing was also conducted with all engineered cell lines and derivatives. Unless specified, experiments were conducted with cells <6 weeks after thawing.

All cells were cultured at 37°C with 5% CO₂. KYSE-520, NCI-H1975, NCI-H1975 CS, LS411N, HCC827, and HCC4006 cells were cultured in RPMI1640 Medium (Thermo Fisher Scientific) supplemented with 10% FBS (Sigma). MIA PaCa-2, MV-4-11, and 293T cells were cultured in high glucose DMEM (Thermo Fisher Scientific) supplemented 10% FBS. MV-4-11-Luc cells were from the Experimental Therapeutics Core at Dana-Farber Cancer Institute (Boston, MA) and cultured in high-glucose DMEM supplemented with 10% FBS and 1 μg/mL puromycin.

The HCC827-ER1 cells that harbor MET amplification (Crown Bioscience UK) were cultured in RPMI1640 medium supplemented with 10% FBS and 42 μmol/L erlotinib (20). The cell line was derived from HCC827 (ATCC), at Crown Bioscience, by culturing the cells in the presence of escalated concentrations of erlotinib. The HCC4006-OsiR cells were generated by culturing HCC4006 cells in the presence of 1 μmol/L osimertinib for approximately 3 months, and were maintained in RPMI1640 medium supplemented with 10% FBS and 1 μmol/L osimertinib.

NCI-H1975 CS cells that harbor EGFR^{L858R/T790M/C797S} mutation were generated through CRISPR Cas9–mediated point mutation at EGFR C797 site, by Synthego Corporation. To generate these cells, ribonucleoproteins containing the Cas9 protein and synthetic, chemically modified single guide RNA were electroporated into the cells using Synthego's optimized protocol. Editing efficiency was assessed upon recovery, 48 hours after electroporation. Genomic DNA was extracted from a portion of the cells, PCR amplified, and sequenced using Sanger sequencing. The resulting chromatograms were processed using Synthego Inference of CRISPR edits software (ice.synthego.com).

KYSE-520 cells stably overexpressing SHP2 WT or SHP2 P491Q were generated by infecting the parental cells with concentrated lentiviruses, and cultured in the media of the parental cells with 1 μg/mL Puromycin (Thermo Fisher Scientific). The cells were split whenever needed, cultured for approximately 2 weeks, and frozen down for future experiments.

In vitro clonogenic assays were conducted with adherent lines plated in 12-well or 24-well plates, treated for 2 weeks, and stained with crystal violet. Ex vivo spheroid proliferation assay was conducted with cells freshly isolated from patient-derived xenograft (PDX), plated in U-bottom ultra-low attachment 96-well plates (Corning) without matrix, and treated for 6 days. Detailed procedures are described in Supplementary Data.

RNA libraries were prepared with the QuantSeq 3′ mRNA-Seq FWD Kit (Lexogen), following the manufacturer's standard protocols. Briefly, libraries were generated with 500 ng total RNA as input and 11 cycles of PCR amplification of the cDNA. Batches of up to 40 samples were multiplexed and each batch was run on NextSeq 500 (Illumina) using the High Output Kit v2 (Illumina).

Sample analyses were conducted using R Bioconductor. Transcript compatibility counts were obtained with kallisto (v0.44.0; ref. 21) running the pseudomode with GENCODE 23 transcript annotations (22). Gene counts were obtained by summing all reads that uniquely mapped, and differential expression analysis was carried out using DESeq2′s (23) default settings. Heatmaps were generated in GraphPad Prism 8.0.

All in vivo work was either approved by the Institutional Animal Care and Use Committee of MD Anderson Cancer Center (Houston, TX) or by the relevant committee of the testing facility. Female mice were used, and body weight was typically 20–28 g when treatment was started.

All subcutaneous models were implanted with 50% Matrigel (Corning). Cell numbers and mouse strains were: KYSE-520, 3 × 10⁶ in NSG mice (The Jackson Laboratory); NCI-H1975, 1 × 10⁶ in CD-1 Nude mice (Charles River Laboratories); and NCI-H1975 CS, 3 × 10⁶ in NSG; and HCC827 and HCC827-ER1, 5 × 10⁶ in athymic nude mice (Envigo). Tumor size was measured with caliper and calculated using a standard formula: length × width²/2. Dosing volume was 10 mL/kg/day. IACS-13909 was formulated in 0.5% methylcellulose, and osimertinib/erlotinib in 0.5% HPMC. For the combination studies, IACS-13909 was dosed in the morning and osimertinib was dosed in the afternoon, with a 6-hour interval in between. To pool tumor measurements from independent experiments, biweekly measurements differing by 1 day across studies were considered as at the same timepoint.

For studies with the MV-4-11 orthotopic model (Experimental Therapeutics Core, Dana-Farber Cancer Institute, Boston, MA), NSG mice were implanted with 2 × 10⁶ MV-4-11-Luc cells (250 μL) intravenously. Mice were enrolled into treatment groups using total flux bioluminescence value, 2 days postimplantation. After dosing ended, all animals were monitored for survival, and euthanized once morbidity and/or stage III paralysis was observed.

Unless specified, data plotting and statistical analysis were conducted using GraphPad Prism 8.0. Graph with error bars represent mean ± SEM.

To discover novel SHP2 inhibitors with drug-like properties, we utilized structure-based design principles starting from known SHP2 allosteric inhibitors, and identified IACS-13909 (Fig. 1A). In an in vitro enzymatic assay, IACS-13909 potently suppressed the phosphatase activity of purified full-length, recombinant human SHP2 protein with an IC₅₀ of approximately 15.7 nmol/L (Fig. 1B). In comparison, in a similar assay using the SHP2 phosphatase domain, IACS-13909 did not suppress phosphatase activity at concentrations up to 50,000 nmol/L, the highest concentration tested (Fig. 1C), suggesting IACS-13909 acts outside the phosphatase domain. The K_d of IACS-13909 binding to SHP2 was approximately 32 nmol/L, as determined by isothermal titration calorimetry analysis (Supplementary Fig. S1A). IACS-13909 is highly selective for SHP2. When tested at 10 μmol/L against a panel of 22 phosphatases, the compound only showed significant inhibition of SHP2 (>50% inhibition; Supplementary Table S1). It is notable that IACS-13909 demonstrated no inhibition of full-length SHP1, the phosphatase that is structurally most similar to SHP2.

To elucidate where IACS-13909 interacts with SHP2 protein, we solved the crystal structure of SHP2_1–530 with IACS-13909 at 2.4 Å resolution with R_free of 0.270 (PDB = 6WU8; Fig. 1D; Supplementary Table S2). The refined structure contained two protomer chains of SHP2_1–530 and two molecules of IACS-13909 in the asymmetric unit. The crystal structure confirmed that the compound bound outside the active site, at the interface between the phosphatase domain (gray) and the C-terminal SH2 domain (cyan), a key allosteric pocket of the protein (24), and stabilizes the inactive state of the enzyme. Key hydrogen bond interactions were observed between the backbone carbonyls of Glu110 and Phe113 and the basic amine group of IACS-13909, as well as between the backbone carbonyl of Glu250 and the pyrazole N–H of the compound. A water molecule bridges between the sidechains of Thr219 and Arg111 and the pyrazine core of the compound and we observed cation-∏ stacking interactions between the Arg111 sidechain and the dichlorobenzene of IACS-13909. Together, these data confirm that IACS-13909 is a direct allosteric inhibitor of SHP2.

Because SHP2 is a critical mediator of oncogenic signaling, a SHP2 inhibitor might be useful as an anticancer agent (2). We evaluated the in vitro antiproliferative effect of IACS-13909 in a panel of 283 cancer cell lines with diverse genomic drivers, using a 10-day two-dimensional proliferation assay. Among the exceptional responder lines (with GI₅₀ ≤ 1 μmol/L), many harbored genetic alterations of RTK or were RTK addicted (sensitive to TKI or RTK short hairpin RNA according to DRIVE; ref. 25). Particularly, all six cell lines with GI₅₀ ≤ 100 nmol/L harbored RTK alterations, DK-MG (EGFR vIII⁺), BV-173 (BCR-ABL), KG-1 (OP2-FGFR1), KU812 (BCR-ABL), SW-13 (ERBB4-IKZF2; ref. 26), and MV-4-11 (FLT3-ITD; Supplementary Fig. S1B). In addition, BRAF V600 mutation appeared to be a predictor of IACS-13909 resistance, with 19 of 23 BRAF V600–mutated cell lines having GI₅₀ > 5 μmol/L. Consistent with the proliferation data, IACS-13909 suppressed pERK in RTK-dependent lines, such as KYSE-520 (EGFR^amp; Supplementary Fig. S1C) and MV-4-11 (FLT3-ITD; Supplementary Fig. S1D), but did not suppress pERK or pMEK in LS411N cells harboring BRAF^V600E (Supplementary Fig. S1E). It is noteworthy that majority of KRAS^mut cell lines in this analysis were resistant to IACS-13909 (Supplementary Fig. S1B), likely due to low coverage of cell lines expressing the nucleotide-cycling KRAS^G12C mutant in this panel and limitations of the two-dimensional culture system in evaluating KRAS^mut cancers. Together, these data demonstrate the antitumor activity of IACS-13909 in cancer cell lines harboring a broad spectrum of activated RTKs, consistent with literature (24).

To ensure that the antiproliferative effect of IACS-13909 in RTK-activated cancer cell lines in vitro was due to inhibition of SHP2, we leveraged the SHP2 proline 491 to glutamine (P491Q) mutant. On the basis of the X-ray crystal structure, Pro491 lines the allosteric binding pocket of SHP2 adjacent to the pyrazolopyrazine ring of IACS-13909. Sequence and structural alignment with SHP1 (PDB = 3PS5) showed glutamine 485 in this position in SHP1 and suggested that a P491Q mutation will abolish IACS-13909 binding to SHP2 due to steric clashes with the glutamine side chain (Fig. 1D), but should still yield a catalytically competent protein (8). Therefore, we stably overexpressed dsRed (control), SHP2 WT, or SHP2 P491Q in the KYSE-520 cells, an EGFR^amp esophageal cancer cell line. Western blotting showed that exogenous SHP2 was expressed at a much higher level than endogenous SHP2 (Fig. 1E). In control cells or cells overexpressing SHP2 WT, IACS-13909 potently suppressed pERK and pMEK levels, but not in cells overexpressing SHP2 P491Q. Similarly, in an in vitro clonogenic assay, whereas IACS-13909 potently suppressed the proliferation of control cells and cells overexpressing SHP2 WT (GI₅₀ <1 μmol/L), overexpression of SHP2 P491Q significantly reduced sensitivity of IACS-13909 (with 7.8-fold shift in IACS-13909 GI₅₀; Fig. 1F). The rescue was specific to SHP2 inhibitor, because EGFRi, erlotinib, demonstrated identical sensitivity in control, SHP2 WT, and SHP2 P491Q-overexpressing cells (Supplementary Fig. S1F and S1G). It is noteworthy that SHP2 P491Q overexpression did not confer complete resistance of KYSE-520 cells to IACS-13909; this is likely because of the presence of endogenous SHP2 in the mutant overexpressing KYSE-520 cells that confers some signaling through the MAPK pathway. Together, these data suggest that IACS-13909 suppresses cell proliferation and signaling through the MAPK pathway in RTK-dependent tumor cells due to its inhibitory effect on SHP2.

To determine the activity of IACS-13909 in vivo, we first evaluated the pharmacokinetic properties of IACS-13909 in mice, rats, and dogs. IACS-13909 demonstrated >70% bioavailability, low clearance rate, and half-lives of ≥7 hours across species, suggesting that the compound is suitable for once per day oral dosing (Supplementary Table S3).

We selected two RTK-dependent cell lines for in vivo evaluation, the EGFR^amp esophageal cancer cell line, KYSE-520, as a representative solid tumor cell line, and the FLT3-ITD⁺ acute myeloid leukemia cell line, MV-4-11, as a representative blood cancer cell line. In mice with established subcutaneous KYSE-520 tumors, IACS-13909 dosed orally at 70 mg/kg once per day potently suppressed tumor growth, with 100% tumor growth inhibition (TGI) observed following 21 days of dosing (Fig. 2A). Importantly, the treatment was well-tolerated, with body weight maintained throughout the study (Fig. 2B). A higher dose of IACS-13909, such as 100 mg/kg once per day, was not tolerated in mice, suggesting 70 mg/kg once per day is approximately the MTD of IACS-13909 in mice.

To confirm that the in vivo antitumor efficacy of IACS-13909 was due to SHP2 inhibition, we analyzed KYSE-520 tumors and blood from mice treated with different dosing levels of IACS-13909. The mRNA levels of DUSP6, an ERK-dependent gene, were used as a readout of SHP2 activity and MAPK pathway signaling in tumors (27). IACS-13909 achieved dose-dependent plasma exposure at 24 hours after a single-dose treatment, and demonstrated dose-dependent suppression of DUSP6 transcript levels in KYSE-520 tumors (Fig. 2C). An inverse correlation between tumor DUSP6 mRNA level and plasma concentration was observed. Among the doses tested, IACS-13909 at 60 or 80 mg/kg span the dose that resulted in tumor stasis in this model (Fig. 2A), maintaining DUSP6 mRNA suppression at >50% throughout the 24-hour dosing interval. These data demonstrate that IACS-13909 potently suppresses MAPK pathway signaling and inhibits growth of an RTK-dependent subcutaneous solid tumor model in vivo.

We further tested the in vivo antitumor efficacy of IACS-13909 in the FLT3-ITD⁺ MV-4-11 leukemia orthotopic model. Mice were implanted with MV-4-11 cells expressing luciferase through tail vein injection, and systemic tumor growth was rapidly established. Mice were randomized on the basis of tumor luminescence levels, and then treated with IACS-13909 at different dosing levels for 5 weeks. Dose-dependent suppression of systemic tumor burden was observed (Fig. 2D and E), with IACS-13909 at 75 mg/kg once per day causing nearly 100% TGI. Importantly, consistent with the suppression of tumor burden, IACS-13909 extended the overall survival of the mice in a dose-dependent manner (Fig. 2F). These data demonstrate the dose-dependent antitumor efficacy of IACS-13909 in an RTK-dependent disseminated leukemia model.

Multiple EGFR TKIs (e.g., erlotinib, gefitinib, and osimertinib) are currently approved in the United States for the first-line treatment of patients with EGFR-activated metastatic NSCLC (13). Most patients on EGFRi treatment will ultimately experience disease progression, with acquired resistance being a major clinical challenge. EGFR mutations in the proximity of the compound binding site (e.g., T790M for erlotinib/gefitinib and C797S or L792H for osimertinib) that preclude drug binding are clinically observed resistance mechanisms (14, 15). Considering that tumors with an EGFR resistance mutation still depend on EGFR and also that SHP2 is a critical mediator of EGFR signaling, we hypothesized that IACS-13909 might have activities in these tumors.

To evaluate the effect of SHP2 inhibition on cancer cells harboring EGFR resistance mutations, we used NSCLC NCI-H1975 cells that harbor both an EGFR-activating mutation (L858R) and resistance mutation (T790M). The NCI-H1975 cells are resistant to erlotinib and sensitive to osimertinib (12). In addition, we generated the NCI-H1975 CS cells, in which EGFR C797S mutation was introduced through the CRISPR Cas9 technology (Supplementary Fig. S2). The NCI-H1975 CS cells demonstrated significantly reduced sensitivity toward osimertinib in an in vitro clonogenic assay, compared with the parental cells (Fig. 3A). Importantly, IACS-13909 potently suppressed the proliferation of both the parental cells and NCI-H1975 CS cells in a dose-dependent manner, with similar potency (GI₅₀ ∼1 μmol/L; Fig. 3B). Consistent with the proliferation data, osimertinib at up to 300 nmol/L failed to suppress the levels of pERK or pEGFR in NCI-H1975 CS cells (Fig. 3C), although in the parental cells, osimertinib at 10 nmol/L was sufficient for potent suppression of pERK and pEGFR (12). Unlike osimertinib, IACS-13909 suppressed pERK in NCI-H1975 CS cells in a dose-dependent manner. As expected, treatment with IACS-13909 did not reduce pEGFR because SHP2 is downstream of EGFR (Fig. 3C). Together, these data demonstrate that IACS-13909 suppresses the proliferation and MAPK pathway signaling in osimertinib-resistant cells harboring an EGFR-dependent resistance mutation in vitro.

To confirm the activity of IACS-13909 in human primary cancer cells, we used the LD1-0025-200717 model, which is a PDX model established from the hydrothorax of a patient with NSCLC who progressed on treatment with osimertinib. The tumor model harbors EGFR^{ex19del/T790M/C797S}, and is resistant to erlotinib, osimertinib, and the combination of the two agents in vivo (28). To rapidly assess the antiproliferative effect of IACS-13909 in this model, we conducted an ex vivo spheroid proliferation assay with cells freshly isolated from tumors grown in mice. As expected, osimertinib treatment at 300 nmol/L, a concentration that is approximately 40-fold higher than the GI₅₀ of osimertinib in NCI-H1975 cells that harbor EGFR^L858R/T790M (Fig. 3A), had little impact on proliferation of the LD1-0025-200717 spheroids ex vivo (Fig. 3D), confirming that this model is resistant to osimertinib. In contrast, IACS-13909 demonstrated dose-dependent suppression of proliferation of the LD1-0025-200717 spheroids ex vivo, with GI₅₀ approximately 1 μmol/L (Fig. 3E), similar to the GI₅₀ of IACS-13909 in the NCI-H1975 cells. These data demonstrate the activity of IACS-13909 in primary cells derived from an osimertinib-resistant EGFR^{ex19del/T790M/C797S} PDX ex vivo.

To determine the in vivo activity of IACS-13909 in tumors harboring an EGFR-dependent resistance mutation, we tested IACS-13909 in the NCI-H1975 parental and NCI-H1975 CS subcutaneous xenograft models in mice. As expected, in the NCI-H1975 parental tumor harboring EGFR^L858R/T790M, erlotinib treatment at 10 mg/kg once per day delivered orally failed to suppress tumor proliferation, and treatment with osimertinib at 5 mg/kg once per day caused regression of the established tumor. Treatment with IACS-13909 at 70 mg/kg once per day demonstrated robust antitumor efficacy, with tumor regression observed (Fig. 3F). In mice bearing the NCI-H1975 CS tumors harboring EGFR^{L858R/T790M/C797S} (Fig. 3G), treatment with osimertinib demonstrated little antitumor efficacy, which was distinct from the response observed in the parental tumors, confirming that this model is resistant to osimertinib in vivo. Importantly, treatment with IACS-13909 at 70 mg/kg once per day also demonstrated robust antitumor efficacy in the NCI-H1975 CS model, with tumor regression observed. Together, these data demonstrate robust activity of IACS-13909 in osimertinib-resistant tumors harboring an EGFR-dependent resistance mutation in vivo.

Beyond EGFR-specific mutations, a major resistance mechanism observed with multiple generations of EGFRis is RTK-bypass, that is, the compensatory activation of alternate RTKs that maintains downstream activation of the MAPK pathway with EGFR inhibited (14, 29–31). Prompted by the antiproliferative effect of IACS-13909 in many RTK-activated human cancer cell lines, we tested the activity of IACS-13909 in EGFR^mut NSCLC cells with RTK-bypass. We generated the HCC4006-osimertinib–resistant (OsiR) model by culturing HCC4006 cells harboring EGFR^ex19del in the presence of 1 μmol/L osimertinib for an extended period of time (∼3 months), and confirmed the reduced osimertinib sensitivity in the OsiR derivative (Fig. 4A). We further demonstrated that the HCC4006-OsiR cells did not harbor resistance mutations in EGFR (i.e., T790M, C797S, etc.) and had decreased pEGFR compared with the parental cells, suggesting that these cells were switching to other oncogenic drivers (Fig. 4B). Indeed, the HCC4006-OsiR cells demonstrated increased expression of multiple RTKs, including FGFR1, Axl, PDGFR, and IGF1Rβ. The OsiR cells had also undergone EMT, with decreased expression of epithelial marker, E-cadherin, and increased expression of mesenchymal markers, vimentin and Zeb1 (Fig. 4B).

We conducted in vitro proliferation assays with HCC4006 parental and HCC4006-OsiR cells, which were treated with IACS-13909 either as a single agent or in combination with osimertinib. Despite the reduced osimertinib sensitivity observed in the OsiR cells, IACS-13909 showed comparable single-agent antiproliferative effect in the parental and OsiR cells in clonogenic assays (Fig. 4C). Importantly, treatment with the combination of IACS-13909 and osimertinib resulted in a synergistic antiproliferative effect in both models (Fig. 4D and E; Supplementary Fig. S3A and S3B), with positive bliss scores in the majority of the concentrations tested (Supplementary Fig. S3C and S3D).

To understand the mechanism of action underlying the antiproliferative effect, signaling analysis was conducted in HCC4006-OsiR cells treated with osimertinib and/or IACS-13909 in vitro. The MPAS (MAPK pathway activity score) signature is composed of 10 genes that reflects MAPK pathway activity (27). On the basis of the MPAS signature, a 13-gene signature (“MPAS-plus”) was developed, which includes three additional MAPK-targeted genes (ETV1, EGR1, and FOSL1; refs. 32, 33). Osimertinib alone failed to potently suppress MAPK pathway signaling in the HCC4006-OsiR cells, as demonstrated by lack of suppression of DUSP6 mRNA levels and other MPAS-plus genes. In contrast, IACS-13909 potently suppressed MAPK pathway signaling, both as a single agent and in combination with osimertinib (Fig. 4F). The suppression was achieved with 2-hour, 48-hour, and 7-day treatment of IACS-13909, suggesting sustained suppression of MAPK pathway, despite the observed partial adaptation (less suppression with prolonged treatment compared with acute treatment). This is consistent with the notion that SHP2 inhibition suppresses the signaling downstream of multiple RTKs, therefore, delaying the multi-RTK–mediated rapid adaptation that is commonly observed toward MAPK pathway inhibitors (34–36). It is also noteworthy that treatment with the combination of IACS-13909 and osimertinib did not cause further suppression of the MAPK pathway compared with IACS-13909 single agent in vitro, suggesting potential additional non-MAPK–mediated mechanisms for the synergistic antiproliferative effect between osimertinib and IACS-13909 in the in vitro setting.

Our in vitro data with IACS-13909, either as single agent or in combination with osimertinib, in the HCC4006-OsiR model that harbors EGFR-independent resistance mechanisms, prompted us to conduct further evaluation in vivo. The osimertinib-resistant EGFR^mut NSCLC HCC827-ER1 cells were generated by exposing HCC827 cells, which harbor an EGFR-activating mutation (EGFR^ex19del), to erlotinib in culture (20). The HCC827-ER1 cells do not harbor EGFR-dependent resistance mutations, but do have amplified c-MET, a genetic alteration observed in tumors from patients who have relapsed on erlotinib and osimertinib (14, 29). The HCC827-ER1 cells are resistant to erlotinib and also to osimertinib (20).

In the osimertinib-sensitive HCC827 xenograft model (Fig. 5A), IACS-13909 at 70 mg/kg dosed daily as a single agent, potently suppressed tumor growth, leading to tumor stasis, and osimertinib dosed as a single agent at 5 mg/kg once per day caused robust tumor regression. As expected, treatment with the combination of IACS-13909 and osimertinib yielded tumor regression, similar to that observed with osimertinib alone, during the period of compound administration. However, following cessation of dosing, tumors in mice treated with the combination did not grow, whereas those treated with osimertinib showed significant growth beginning approximately 30 days after the final dose. Importantly, the combination treatment in HCC827 xenograft model was tolerated, as shown by the maintenance of body weight during the study (<10% average body weight loss; Supplementary Fig. S4A). Thus, treatment with the combination of IACS-13909 and osimertinib resulted in a more durable antitumor response (Fig. 5A), consistent with the in vitro observation in the HCC4006 cells (Fig. 4D).

In the osimertinib-resistant HCC827-ER1 model (Fig. 5B), tumors in mice treated with IACS-13909 at 70 mg/kg once per day demonstrated tumor stasis, similar to the response in the HCC827 parental model. However, tumors in mice treated with osimertinib continued to grow on treatment, indicating reduced osimertinib sensitivity in HCC827-ER1 as compared with the HCC827 model. Importantly, the combination treatment of IACS-13909 and osimertinib caused robust regression of the HCC827-ER1 tumor, similar to the single-agent effect of osimertinib in the parental model. Waterfall plot of tumor volume demonstrated that almost all mice treated with the combination for 3 weeks had tumor regression (Fig. 5C). The combination of osimertinib and IACS-13909 in the HCC827-ER1 xenograft model was also tolerated, as shown by the maintenance of body weight during the study (≤5% body weight loss; Supplementary Fig. S4B). This result demonstrates that addition of the SHP2 inhibitor, IACS-13909, leads to resensitization of the osimertinib-resistant HCC827-ER1 model harboring RTK-bypass to treatment with osimertinib.

We further determined whether combination of osimertinib and IACS-13909 can inhibit the growth of HCC827-ER1 tumors that progressed on osimertinib treatment. We started osimertinib single-agent treatment when average tumor volume was approximately 300 mm³; the tumors progressed and average tumor volume reached approximately 500 mm³ (67% increase in tumor volume) within 3 weeks. At this time, the osimertinib-treated mice were rerandomized and enrolled into treatment with osimertinib alone or with the combination of osimertinib and IACS-13909. Whereas tumors in mice treated with osimertinib alone continued to grow, tumors in mice treated with the combination of osimertinib and IACS-13909 demonstrated significantly inhibited tumor growth, with tumor regression (Fig. 5D).

To gain insight into the mechanism of the combination treatment, we analyzed HCC827-ER1 tumors from mice treated with osimertinib alone, IACS-13909 alone, or the combination. Mice were dosed following the same schedule as in the efficacy study, with a 6-hour interval between IACS-13909 dosing and osimertinib dosing (Fig. 5E), and samples were harvested at three timepoints during the 24-hour dosing cycle. In the HCC827-ER1 tumors, osimertinib treatment modestly suppressed DUSP6 mRNA levels. IACS-13909 potently suppressed DUSP6 mRNA levels at 6- and 8-hour after treatment, with the extent of suppression decreased afterwards. Importantly, the combined treatment with the two compounds maintained the potent suppression of DUSP6 mRNA levels throughout the 24-hour dosing cycle (Fig. 5E). To further confirm the combinational effect on MAPK pathway signaling, we evaluated the MPAS-plus panel (Fig. 5F). At close to dosing trough, while each monotherapy had little effect on the mRNA levels of the genes on the panel, the combination of IACS-13909 and osimertinib more potently suppressed the level of most of the MPAS-plus genes. These data suggest that combined treatment with osimertinib and IACS-13909 potently suppresses MAPK pathway signaling, to a larger extent than either single agent, in an osimertinib-resistant model with RTK-bypass, consistent with the efficacy data.

In this study, we report the discovery of IACS-13909, a potent and selective allosteric SHP2 inhibitor. Our in vitro and in vivo data demonstrate that IACS-13909 has antitumor activity and suppresses MAPK pathway signaling in RTK-dependent cancers. Importantly, IACS-13909 exhibits antitumor efficacy in osimertinib-resistant models that harbor clinically relevant resistance mechanisms. In osimertinib-resistant tumors with EGFR-dependent resistance mutations, such as the C797S mutation in the NCI-H1975 CS cells, EGFR remains the primary oncogenic driver and signals through SHP2. Thus, although osimertinib is not able to potently suppress EGFR here, inhibition of SHP2, which lies downstream of EGFR, blocks signaling through the MAPK pathway (Fig. 6A). In osimertinib-resistant tumors, in which the MAPK pathway is activated because of activation of an alternate RTK, such as MET in the HCC827-ER1 cells, the alternate RTK signals through SHP2 to maintain the MAPK pathway activity (Fig. 6B). Together, our preclinical data demonstrate that the SHP2 inhibitor, IACS-13909, is effective in overcoming both EGFR-dependent and EGFR-independent resistance mechanisms toward osimertinib. Importantly, the ability of SHP2 inhibition in targeting multiple resistance mechanisms is anticipated to address the heterogeneity and plasticity of osimertinib resistance.

A major challenge in targeting the RTK/MAPK pathway is acquired resistance, whereby a tumor initially responds to treatment, but regrows on continued treatment. This can be attributed to both the adaptability of the cancer cells and the heterogeneity of the primary tumors. Cancer cells very often harbor a primary oncogenic driver. When the primary driver is blocked, other oncogenic drivers either within the same cells or from a different clone emerge as the alternate driving force for tumor growth. Combining one drug targeting the primary oncogenic driver and a second drug suppressing multiple potential alternative drivers is an attractive strategy. To improve the therapeutic index, ideally the first drug should be mutant selective, and to ensure broad targeting of potential secondary drivers, the second drug should target WT protein as well. Here, we propose combining EGFR-mutant selective inhibitor, osimertinib (12), and SHP2 allosteric inhibitor, IACS-13909, that is not mutant selective. At tolerated doses, such combination achieves more durable response compared with osimertinib single agent in osimertinib-sensitive EGFR^mut NSCLC tumors, and causes tumor regression in osimertinib-resistant EGFR^mut NSCLC xenograft tumors in mice.

Our data demonstrate that IACS-13909 has antitumor activity in cancers with a broad range of RTKs as the oncogenic driver. While we provide data showing SHP2 inhibition can overcome both EGFR-dependent and EGFR-independent osimertinib resistance in EGFR^mut NSCLC, a SHP2 inhibitor can be used more broadly. Several additional combination strategies with an allosteric SHP2 inhibitor have been proposed in overcoming resistance to targeted agents. First, in ALK inhibitor–resistant preclinical models with RTK-bypass as a resistance mechanism, in vivo efficacy for the treatment with combination of ALK inhibitor, ceritinib, and SHP099 has been reported (37). Second, the clinical response to MEK inhibitors is limited by adaptive feedback activation through multiple RTKs (35, 38), therefore, combination of SHP2 inhibitor and MEK inhibitor has demonstrated antitumor efficacy in mice (8, 36). In addition to MEK inhibitor, preclinical data for combining ERK inhibitor have been reported (39). A major challenge with combining SHP2 inhibitor and MEK inhibitor (or ERK inhibitor) when both molecules target WT enzymes is the therapeutic index. SHP2 inhibitors were used at reduced doses or dosing frequencies in both combination strategies in mice (36, 39). It is speculated that sustained shutdown of the MAPK pathway in normal tissue may not be tolerated, therefore, reduced dose or dosing frequency that leads to pulsatile shutdown of the MAPK pathway had to be performed. Most recently, multiple approaches have identified combining a KRAS^G12C-mutant–specific inhibitor and SHP2 inhibitor as a strategy for achieving more robust and durable response (40, 41). Identifying the most optimal combination strategy for a SHP2 allosteric inhibitor requires additional preclinical work and, most importantly, clinical trials. Currently, several SHP2 allosteric inhibitors (TNO155, RMC-4630, JAB-3068, JAB-3312, and RLY-1971) are under early-phase clinical development. An advanced derivative of IACS-13909 will enter phase I clinical trial in later 2020.

Y. Sun reports other funding from Navire Pharma (this work was funded by Navire Pharma Inc., a BridgeBio Company. The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study. B.A. Meyers reports other funding from Navire Pharma Inc. (this work was funded by Navire Pharma Inc., a BridgeBio Company. The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study. B. Czako reports other funding from Navire Pharma (this work was funded by Navire Pharma Inc., a BridgeBio Company. The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study, as well as has a patent for WO 2020033828 A1 20200213 issued and a patent for US 20170342078 A1 20171130 issued. P. Leonard reports other funding from Navire Pharma Inc. (this work was funded by Navire Pharma Inc., a BridgeBio Company. The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study, as well as has a patent for US 10280171 issued and licensed to Navire Pharma Inc. F. Mseeh reports other funding from Navire Pharma Inc. (this work was funded by Navire Pharma Inc., a BridgeBio Company. The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study, as well as has a patent for US 10280171 issued and licensed to Navire Pharma Inc. C.A. Parker reports other funding from Navire Pharma Inc. (this work was funded by Navire Pharma Inc., a BridgeBio Company. The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study, as well as has a patent for US10280171B2 issued. J.B. Cross reports other funding from Navire Pharma Inc. (this work was funded by Navire Pharma Inc., a BridgeBio Company. The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study, as well as has a patent for WO2020033828 pending and licensed to Navire Pharma and a patent for US 10280171 issued and licensed to Navire Pharma. M.E. Di Francesco reports other funding from Navire Pharma Inc. (this work was funded by Navire Pharma Inc., a BridgeBio Company. The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study and personal fees from ImmunoGenesis, Inc. (consultant for organic synthesis and medicinal chemistry) outside the submitted work. B.J. Bivona reports other funding from Navire Pharma Inc. (this work was funded by Navire Pharma Inc., a BridgeBio Company. The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study. C.A. Bristow reports other funding from Navire Pharma Inc. (this work was funded by Navire Pharma Inc., a BridgeBio company. The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study. J.P. Burke reports other funding from MD Anderson (this work was funded by Navire Pharma Inc., a BridgeBio Company. The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study and MD Anderson (salary paid during prior time until July 2018 when no longer employee) outside the submitted work, as well as has a patent for WO2019213318 A1 issued (inventor on relevant patent). C.L. Carroll reports other funding from Navire Pharma Inc., a BridgeBio Company (this work was funded by Navire Pharma Inc., a BridgeBio Company. The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study, as well as has a patent for WO/2020/033828 issued to Board of Regents, The University of Texas System. G. Gao reports other funding from Navire Pharma Inc. (this work was funded by Navire Pharma Inc., a BridgeBio Company. The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study. S. Gera reports other funding from Navire Pharma Inc. (this work was funded by Navire Pharma Inc., a BridgeBio Company. The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study. V. Giuliani reports other funding from Navire Pharma Inc., a BridgeBio Company (the University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study. J.K. Huang reports grants, nonfinancial support, and other funding from Navire Pharma Inc. (this work was funded by Navire Pharma Inc., a BridgeBio Company. The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire) during the conduct of the study. J.J. Kovacs reports other funding from Navire Pharma (this work was funded by Navire Pharma Inc., a BridgeBio Company. The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study. C.-Y. Liu reports other funding from Navire Pharma (this work was funded by Navire Pharma Inc., a BridgeBio Company. The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study. A.M. Lopez reports other funding from Navire Pharma Inc. (this work was funded by Navire Pharma Inc., a BridgeBio Company. The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study. X. Ma reports other funding from Navire Pharma Inc., a BridgeBio Company (The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study. T. McAfoos reports other funding from Navire Pharma Inc. (this work was funded by Navire Pharma Inc., a BridgeBio Company. The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study, as well as has a patent for WO 2019213318 issued. M.A. Miller reports other funding from Navire Pharma (this work was funded by Navire Pharma Inc., a BridgeBio Company. The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study. M. Peoples reports other funding from Navire Pharma (this work was funded by Navire Pharma Inc., a BridgeBio Company. The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study. V. Ramamoorthy reports other funding from Navire Pharma (this work was funded by Navire Pharma Inc., a BridgeBio Company. The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study. N.D. Spencer reports other funding from Navire Pharma Inc. (this work was funded by Navire Pharma Inc., a BridgeBio Company. The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study. E. Suzuki reports other funding from Navire Pharma (this work was funded by Navire Pharma Inc., a BridgeBio Company. The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study. C.C. Williams reports other funding from Navire Pharma (this work was funded by Navire Pharma Inc., a BridgeBio Company. The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study. A.M. Zuniga reports other funding from Navire Pharma (this work was funded by Navire Pharma Inc., a BridgeBio Company. The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study. G.F. Draetta reports other funding from Navire Pharma (this work was funded by Navire Pharma Inc., a BridgeBio Company. The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study and personal fees from Taiho Pharmaceutical Co., Blueprint Medicines, BiovelocITA (stock ownership), Metabomed (stock ownership), Nurix, Inc. (stock ownership), Orionis Biosciences (stock ownership), Frontier Medicines (stock ownership), and Forma Therapeutics (stock ownership) outside the submitted work. J.R. Marszalek reports other funding from Navire Pharma (sponsored research) during the conduct of the study. T.P. Heffernan reports other funding from Navire Pharma Inc. (this work was funded by Navire Pharma Inc., a BridgeBio Company. The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study and personal fees and other compensation from Cullgen Inc. (stock ownership and advisory fees) outside the submitted work. P. Jones reports other funding from Navire Pharma Inc (this work was funded by Navire Pharma Inc., a BridgeBio Company. The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaboration and license pursuant to which MD Anderson and Navire will collaborate on the conduct of research and development of products. Under this agreement, the Board of Regents of The University of Texas System, on behalf of MD Anderson, received equity, milestone payments, and royalties in Navire. Proceeds may be distributed based on UT System Intellectual Property policy) during the conduct of the study, as well as has a patent for WO2020033828 pending and licensed to Navire Pharma Inc. and a patent for US 10280171 issued and licensed to Navire Pharma Inc. No potential conflicts of interest were disclosed by the other authors.

Y. Sun: Conceptualization, data curation, formal analysis, supervision, validation, investigation, visualization, methodology, writing-original draft, project administration, writing-review and editing. B.A. Meyers: Data curation, formal analysis, validation, investigation, methodology. B. Czako: Conceptualization, supervision, investigation, project administration, writing-review and editing. P. Leonard: Formal analysis, supervision, investigation, visualization, methodology, writing-original draft, writing-review and editing. F. Mseeh: Supervision, methodology, writing-original draft. A.L. Harris: Validation, investigation, methodology. Q. Wu: Validation, investigation, methodology. S. Johnson: Validation, investigation, methodology. C.A. Parker: Validation, investigation, methodology. J.B. Cross: Supervision, writing-review and editing. M.E. Di Francesco: Resources, supervision. B.J. Bivona: Methodology. C.A. Bristow: Formal analysis, supervision. J.P. Burke: Investigation. C.C. Carrillo: Investigation. C.L. Carroll: Investigation. Q. Chang: Investigation, methodology. N. Feng: Supervision. G. Gao: Investigation. S. Gera: Investigation. V. Giuliani: Supervision. J.K. Huang: Formal analysis, methodology, writing-original draft. Y. Jiang: Supervision, writing-original draft. Z. Kang: Investigation. J.J. Kovacs: Supervision, writing-review and editing. C.-Y. Liu: Investigation. A.M. Lopez: Methodology. X. Ma: Investigation. P.K. Mandal: Investigation. T. McAfoos: Investigation. M.A. Miller: Investigation. R.A. Mullinax: Investigation. M. Peoples: Supervision, investigation. V. Ramamoorthy: Investigation. S. Seth: Data curation, investigation. N.D. Spencer: Investigation. E. Suzuki: Investigation. C.C. Williams: Investigation. S.S. Yu: Investigation. A.M. Zuniga: Methodology. G.F. Draetta: Resources, supervision, funding acquisition. J.R. Marszalek: Resources, supervision. T.P. Heffernan: Resources, supervision, writing-review and editing. N.E. Kohl: Conceptualization, resources, supervision, project administration, writing-review and editing. P. Jones: Conceptualization, resources, supervision, funding acquisition, project administration, writing-review and editing.

The authors thank DV-MS at MD Anderson Cancer Center (MDACC) for mouse husbandry and care, Research Histology, Pathology & Imaging Core at MDACC Science Park for histology service, and all members at TRACTION and the Institute for Applied Cancer Science at MDACC for discussions. The authors also thank ChemPartners, Eurofins, Crown Bioscience, LIDE Biotech, and the experimental therapeutics core at the Dana-Farber Cancer Institute for service.

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