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Supplementary Table 4: List of cell lines evaluated for sensitivity to SHP099 (Tab 1) or an RTK-inhibitor (Tab 2) in 2D or 3D. Included is the lineage and the KRAS mutation status. Where data are blank, no viable data was available, or the cell line did not grow appropriately as a tumor spheroid.

Supplementary Table 3: A total of 246 cell lines were evaluated for both SHP2 knockdown and SHP099 sensitivity. Data displays PTPN11 shRNA ATARIS Quantile Score and SHP099 IC50 and Amax for each cell line tested.

In vivo efficacy of SHP099 (100 mg/kg, daily) in the KYSE-520 esophageal cancer cell line model. Data are plotted as the treatment mean {plus minus} s.e.m (n=7) ( (B-I) In vivo efficacy data for cell line models represented in Fig. 3E. SHP099 and trametinib were orally administered at the doses, schedules and for the duration indicated. Data are plotted as the treatment mean {plus minus} s.e.m.

(A) Immunoblot of p-RSK3 and qPCR for DUSP6 from MIA PaCa-2 xenografts collected 3 hours after the last dose from Fig 5C. (B) Immunoblot for the designated proteins from MIA PaCa-2 cells grown in 2D, 3D and from in vivo xenografts without compound treatment. Protein loading amount was normalized and verified by tubulin loading control. Each separate column represents an individual treated tumor. (C) Dependency of MET by DRIVE pooled shRNA screen (y axis, ATARIS Quantile score of less than -0.5 indicates a significant effect) and expression of HGF (x-axis) by RNAseq in pancreatic cancer cell lines in CCLE (n=21). (D) Immunoblot of p-RSK3 and qPCR for DUSP6 from KP4 xenografts collected 3 hours after the last dose from Fig 5E. Protein loading amount was normalized and verified by tubulin loading control. Each separate column represents an individual treated tumor (E) Immunoblot for the designated proteins from KP4 cells grown in 2D, 3D and from in vivo xenografts without compound treatment (F) Schematic of RTK-SHP2 signaling highlighting that SHP2 acts downstream of one or more activated RTKs to elicit downstream signaling in KRAS mutant and also additional SHP2-specific, non-MAPK signaling. SHP2 inhibition by SHP099 can serve as a surrogate for cancers where KRAS mutant cancers are dependent on upstream RTKs.

Supplementary Table 1: List of all cell lines in the CCLE (Cancer Cell Line Encyclopedia) where sensitivity to SHP2 (PTPN11) knockdown was evaluated in 2D. Lineage, and genetic status of KRAS and BRAF are shown. Blank cells represent cell line data where genetic or sensitivity data do not exist. Values depicted are the ATARIS quantile normalized z-score. Additional details on the methodology of the screen are published (58).

(A) Effects of SHP099 and erlotinib on proliferation of EGFR-dependent and RAS/RAF wildtype Detroit-562 and KYSE520 cells grown in 2D monolayer or 3D spheroids (with 20% matrigel) for 6 days. The colored dotted lines at the bottom of graphs are the percentage of Day 0 reading of DMSO-treated cells normalized to that of Day 6 in 2D and 3D, respectively. The black hyphenated lines visualize IC50 values. Error bars denote standard error of the mean. (B-C) Immunoblot of indicated proteins in T3M-4 cells treated with DMSO, SHP099 (10 µM), or trametinib (10 nM) for 2 hours in 2D and 3D culture, respectively. Tubulin serves as a protein loading control. (D) Heatmap depicts Log2 fold change of genes differentially regulated in 2D and 3D following 19 hours after treatment with SHP099. Duplicate samples were analyzed and are depicted. GO ontology was performed using DAVID (42,43) derived from genes with 2-fold change relative to DMSO control.

Supplementary Material and Methods. File contains the following: Transcriptome sequencing and analysis, Soft agar assay, 2D and 3D Cell proliferation screen and compound characterization information.

Supplementary Table 2: List of all cell lines in the CCLE where sensitivity to SHP099 was evaluated in 2D. Lineage and genetic status of KRAS and BRAF are shown. Blank cells represent cell line data where genetic or screening data do not exist. Data depicted in Figure 1B are IC50 values. This table includes all sensitivity data based on both IC50 and Amax data.

(A) In vivo primary human colorectal xenograft model HCOX4087 treated with trametinib (0.3 mg/kg QD), SHP099 (100 mg/kg QD), and a pan-RTK inhibitor, Dovitinib (100 mg/kg QD). (B-C) In vivo efficacy of selective VEGFR2 inhibitor, BFH772 (3 mg/kg QD) in MIA PaCA-2 and T3M-4 cells implanted subcutaneously. Data plotted are tumor volume means {plus minus} s.e.m (T3M-4, n=8. MIA PaCA-2, n=7) (D) Evaluation of SHP099 in firefly- luciferase labeled MIA PaCa-2 pancreatic cells implanted surgically into the mouse pancreas. Data plotted are mean bioluminescent signal (BLI) {plus minus} s.e.m (n=6). (E-F) Immunoblot of SHP2 and soft agar assay with MIA Paca-2 cells with Dox-inducible shRNA targeting SHP2 or control non-targeting shRNA after Dox treatment. (G) In vivo SHP2 expression levels evaluated by Western blot and levels of MAPK pathway suppression by DUSP6, 3 hours after the last dose of SHP099 from MIA PaCa-2 tumors in Fig. 4F (n=3).

Purpose: SHP2 inhibitors offer an appealing and novel approach to inhibit receptor tyrosine kinase (RTK) signaling, which is the oncogenic driver in many tumors or is frequently feedback activated in response to targeted therapies including RTK inhibitors and MAPK inhibitors. We seek to evaluate the efficacy and synergistic mechanisms of combinations with a novel SHP2 inhibitor, TNO155, to inform their clinical development. Experimental Design: The combinations of TNO155 with EGFR inhibitors (EGFRi), BRAFi, KRASG12Ci, CDK4/6i, and anti–programmed cell death-1 (PD-1) antibody were tested in appropriate cancer models in vitro and in vivo, and their effects on downstream signaling were examined. Results: In EGFR-mutant lung cancer models, combination benefit of TNO155 and the EGFRi nazartinib was observed, coincident with sustained ERK inhibition. In BRAFV600E colorectal cancer models, TNO155 synergized with BRAF plus MEK inhibitors by blocking ERK feedback activation by different RTKs. In KRASG12C cancer cells, TNO155 effectively blocked the feedback activation of wild-type KRAS or other RAS isoforms induced by KRASG12Ci and greatly enhanced efficacy. In addition, TNO155 and the CDK4/6 inhibitor ribociclib showed combination benefit in a large panel of lung and colorectal cancer patient–derived xenografts, including those with KRAS mutations. Finally, TNO155 effectively inhibited RAS activation by colony-stimulating factor 1 receptor, which is critical for the maturation of immunosuppressive tumor-associated macrophages, and showed combination activity with anti–PD-1 antibody. Conclusions: Our findings suggest TNO155 is an effective agent for blocking both tumor-promoting and immune-suppressive RTK signaling in RTK- and MAPK-driven cancers and their tumor microenvironment. Our data provide the rationale for evaluating these combinations clinically.

KRAS, an oncogene mutated in nearly one third of human cancers, remains a pharmacologic challenge for direct inhibition except for recent advances in selective inhibitors targeting the G12C variant. Here, we report that selective inhibition of the protein tyrosine phosphatase, SHP2, can impair the proliferation of KRAS-mutant cancer cells in vitro and in vivo using cell line xenografts and primary human tumors. In vitro, sensitivity of KRAS-mutant cells toward the allosteric SHP2 inhibitor, SHP099, is not apparent when cells are grown on plastic in 2D monolayer, but is revealed when cells are grown as 3D multicellular spheroids. This antitumor activity is also observed in vivo in mouse models. Interrogation of the MAPK pathway in SHP099-treated KRAS-mutant cancer models demonstrated similar modulation of p-ERK and DUSP6 transcripts in 2D, 3D, and in vivo, suggesting a MAPK pathway–dependent mechanism and possible non-MAPK pathway–dependent mechanisms in tumor cells or tumor microenvironment for the in vivo efficacy. For the KRASG12C MIAPaCa-2 model, we demonstrate that the efficacy is cancer cell intrinsic as there is minimal antiangiogenic activity by SHP099, and the effects of SHP099 is recapitulated by genetic depletion of SHP2 in cancer cells. Furthermore, we demonstrate that SHP099 efficacy in KRAS-mutant models can be recapitulated with RTK inhibitors, suggesting RTK activity is responsible for the SHP2 activation. Taken together, these data reveal that many KRAS-mutant cancers depend on upstream signaling from RTK and SHP2, and provide a new therapeutic framework for treating KRAS-mutant cancers with SHP2 inhibitors.

FGFR1 was recently shown to be activated as part of a compensatory response to prolonged treatment with the MEK inhibitor trametinib in several KRAS-mutant lung and pancreatic cancer cell lines. We hypothesize that other receptor tyrosine kinases (RTK) are also feedback-activated in this context. Herein, we profile a large panel of KRAS-mutant cancer cell lines for the contribution of RTKs to the feedback activation of phospho-MEK following MEK inhibition, using an SHP2 inhibitor (SHP099) that blocks RAS activation mediated by multiple RTKs. We find that RTK-driven feedback activation widely exists in KRAS-mutant cancer cells, to a less extent in those harboring the G13D variant, and involves several RTKs, including EGFR, FGFR, and MET. We further demonstrate that this pathway feedback activation is mediated through mutant KRAS, at least for the G12C, G12D, and G12V variants, and wild-type KRAS can also contribute significantly to the feedback activation. Finally, SHP099 and MEK inhibitors exhibit combination benefits inhibiting KRAS-mutant cancer cell proliferation in vitro and in vivo. These findings provide a rationale for exploration of combining SHP2 and MAPK pathway inhibitors for treating KRAS-mutant cancers in the clinic.

Background: WNT974, a Porcupine inhibitor, has shown evidence of Wnt pathway inhibition in clinical trials. Dysregulated Wnt signaling has been linked to immunotherapy resistance, suggesting WNT974 may act synergistically with checkpoint inhibitors. Spartalizumab is an αPD-1 mAb with demonstrated clinical activity in solid tumors. Methods: In this Phase I, open-label trial (NCT01351103) adult pts received WNT974 ± spartalizumab; here we report on the dose escalation of the combination. Eligible pts had melanoma (including uveal), lung SCC, HNSCC, esophageal SCC, cervical SCC, or TNBC. Pts with melanoma, lung SCC, or HNSCC must have had a best response of progressive disease (primary refractory) to prior αPD-1 therapy; other pts were naïve or primary refractory to prior αPD-1. WNT974 was dosed orally QD in 28-day cycles (2.5-10 mg, Days 1-8 or 1-15 of Cycles 1 or 1-4); spartalizumab was dosed IV at 400 mg Q4W. Objectives were to determine the maximum tolerated dose (MTD)/recommended dose for expansion (RDE), safety, pharmacokinetics (PK), pharmacodynamics, and activity of WNT974 + spartalizumab. Pre- and on-treatment pt samples were collected: skin samples for RT-PCR analysis of AXIN2, a marker of Wnt pathway activity; tumor samples for RNAseq of AXIN2 and immune cell markers. Results: As of Sept 2, 2019, 27 pts were enrolled: 24 discontinued (18 due to disease progression; 67%), 3 were ongoing. Most common tumor types were non-uveal melanoma (n=8), TNBC (n=7), and uveal melanoma (n=5); 63% had received prior αPD-1. PK parameters for WNT974 + spartalizumab were consistent with prior single agent data. Dose-limiting toxicities were reported in 2 pts: Grade (G) 2 spinal compression fracture that occurred in the setting of trauma and G3 arthralgia. 78% of pts experienced a treatment-related AE, the most common being hypothyroidism (19%); 4 pts (15%) had 7 suspected-related G3/4 AEs (arthralgia, atrial fibrillation, diabetes mellitus, diabetic ketoacidosis, hyperglycemia, hyponatremia, and maculopapular rash). One pt (4%) with TNBC had a partial response, 11 pts (41%) had stable disease (SD), 13 pts (48%) had progressive disease; response was unknown in 2 pts. SD was reported in 9/17 pts (53%) who were primary refractory to prior αPD-1; 4 remained on study >24 wks. All pts with uveal melanoma (n=5) had SD. Evidence of Porcupine inhibition, assessed by skin AXIN2 suppression, was detected at all dose levels studied. Pts with the largest reductions in tumor size had on-treatment increases in immune marker mRNA in tumor samples, including a pt with αPD-1 primary refractory melanoma with high baseline AXIN2 expression and 42% reduction in the sum of target lesion diameters; this pt remained on study at 48 wks at the cutoff date. Conclusions: WNT974 + spartalizumab was well tolerated; MTD/RDE have not been determined. Preliminary data suggest blocking Wnt signaling may enable response to checkpoint inhibition in some pts. Citation Format: Filip Janku, Filip de Vos, Maria de Miguel, Patrick Forde, Antoni Ribas, Misako Nagasaka, Guillem Argiles, Ana Maria Arance, Aitano Calvo, Marios Giannakis, Maritza Melendez, Jiachang Gong, Sebastian Szpakowski, Rebecca Kan, Susan E. Moody, Maja De Jonge. Phase I study of WNT974 + spartalizumab in patients (pts) with advanced solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT034.

Introduction: The RTK-RAS-MAPK pathway is frequently activated in cancers due to a variety of mechanisms including mutations or amplifications in RTK, KRAS or BRAF. The effectiveness of inhibitors targeting those oncogenic drivers is often limited by the pathway feedback activation originated at the RTK level in response to ERK inhibition. The non-receptor protein tyrosine phosphatase SHP2 mediates RAS activation downstream of various receptor tyrosine kinases. Potent and selective allosteric SHP2 inhibitors such as TNO155 are in clinical development and offer an appealing one-size-fits-all approach to overcome RTK-mediated feedback activation of RAS and enhance the efficacy of inhibitors targeting RTK, BRAF or KRASG12C. We sought to study the combination efficacy and mechanism in pre-clinical models for prioritization in clinical trials. Experimental procedures: The combination efficacy and synergy of TNO155 with EGF816 (a 3rd generation EGFR inhibitor), dabrafenib+trametinib or a tool KRAS G12C inhibitor (G12Ci) were respectively assessed in a number of EGFR mutant lung cancer models, BRAFV600E colorectal cancer models and KRASG12C lung and colorectal cancer models. The effect of the combinations on ERK inhibition was also studied. Results: TNO155 has varying single agent activity in vitro in EGFR mutant lung cancer cell lines and is not affected by clinically relevant resistance mechanisms to EGFR inhibitors such as secondary mutations in EGFR (T790M and C797S) or MET amplification. In addition, the combination of TNO155 and EGF816 is synergistic across cell lines, coincident with sustained ERK inhibition. In BRAFV600E colorectal cancer cell lines with feedback activation of EGFR, MET or FGFR respectively in response to treatment with dabrafenib+trametinib, TNO155 synergistically enhanced the efficacy of dabrafenib+trametinib in all three cell lines, phenocopying respective RTK inhibitors. In KRASG12C lung cancer cell lines, quick rebound of p-ERK was observed as early as 24 hour post treatment with G12Ci and cannot be blocked by increasing concentrations of G12Ci, suggesting feedback activation of wild-type KRAS or other RAS isoforms. In contrast, TNO155 effectively blocked the p-ERK rebound and enhanced the efficacy of G12Ci. Similar observations were made in KRASG12C colorectal cancer cell lines. Conclusions: Our findings suggest that SHP2 inhibition is an effective strategy to block the feedback activation of wild type and G12C KRAS, as well as NRAS and HRAS, by a variety of RTKs, in response to ERK inhibition. Given the mutant selective properties of those inhibitors we studied, the tolerability of their combinations with TNO155 is highly expected. Our data provide pre-clinical rationale to explore those TNO155 combinations in the corresponding cancer indications in clinic. Citation Format: Huaixiang Hao, Chen Liu, Hongyun Wang, Hengyu Lu, Scott Delach, Matthew LaMarche, Jeffrey Engelman, Peter Hammerman, Giordano Caponigro, Susan Moody, Morvarid Mohseni. Combinations of SHP2 inhibitor to overcome RAS activation by receptor tyrosine kinases in response to ERK inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-122.

FGFR1 was recently shown to be activated as part of a compensatory response to prolonged treatment with MEK inhibitor trametinib in several KRAS-mutant lung and pancreatic cancer cell lines. We hypothesize that other receptor tyrosine kinases (RTKs) are also feedback activated in this context. Herein, we profile a large panel of KRAS-mutant cancer cell lines for the contribution of RTKs to the feedback activation of phospho-MEK following MEK inhibition, using a SHP2 inhibitor (SHP099) that blocks RAS activation mediated by multiple RTKs. We find that RTK-driven feedback activation widely exists in KRAS mutant cancer cells and involves several RTKs including EGFR, FGFR, and MET. We further demonstrate this pathway feedback activation is mediated through mutant KRAS. Finally, SHP099 and MEK inhibitors exhibit combination benefits inhibiting KRAS mutant cancer cell proliferation in vitro and in vivo. These findings provide a rationale for exploration of combining SHP2 and MAPK pathway inhibitors for treating KRAS-mutant cancers in the clinic. Citation Format: Hengyu Lu, Chen Liu, Roberto Velazquez, Hongyun Wang, Lukas M. Dunkl, Malika Kazic-Legueux, Anne Haberkorn, Eric Billy, Eusebio Manchado, Saskia M. Brachmann, Susan Moody, Jeffrey A. Engelman, Peter S. Hammerman, Giordano Caponigro, Morvarid Mohseni, Huai-Xiang Hao. SHP2 inhibition overcomes RTK-mediated pathway reactivation in KRAS-mutant tumors treated with MEK inhibitors [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A44.

Introduction: FGFR1 was recently shown to be activated as part of a compensatory response to prolonged treatment with MEK inhibitor (MEKi) such as trametinib in several KRAS mutant lung and pancreatic cancer cell lines. We hypothesize that other receptor tyrosine kinases (RTKs) are also feedback activated in KRAS mutant cell lines after MEKi treatment. Experimental procedures: We profiled a large panel (n>32) of KRAS mutant cancer cell lines for the contribution of RTKs to the feedback activation of phospho-MEK following MEK inhibition, using a SHP2 inhibitor (SHP099) that blocks RAS activation mediated by multiple RTKs. We then performed in vitro and in vivo combination efficacy studies and pathway analysis in various KRAS mutant cancer models. Results: We find that RTK-driven feedback activation widely exists in KRAS mutant cancer cells and involves several RTKs including EGFR, FGFR, and MET. We further demonstrate that this pathway feedback activation is mediated through mutant KRAS in KRAS G12C or G12D models. Finally, SHP099 and MEK inhibitors exhibit combination benefits inhibiting MAPK pathway and KRAS mutant cancer cell proliferation in vitro and in vivo. Conclusions: Our findings suggest that MAPK inhibition in KRAS mutant cancer provokes feedback re-activation of the pathway that often involves RTK activity and SHP2 inhibition may enhance the efficacy of MEKi in KRAS mutant tumors. These findings provide a rationale for exploration of combining SHP2 and MAPK pathway inhibitors for treating KRAS mutant cancers in the clinic. Citation Format: Hengyu Lu, Chen Liu, Roberto Velazquez, Hongyun Wang, Lukas M. Dunkl, Malika Kazic-Legueux, Anne Haberkorn, Eric Billy, Eusebio Manchado, Saskia M. Brachmann, Susan Moody, Jeffrey A. Engelman, Peter S. Hammerman, Giordano Caponigro, Morvarid Mohseni, Huaixiang Hao. SHP2 inhibition overcomes RTK-mediated pathway reactivation in KRAS mutant tumors treated with MEK inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 954.

Amplification and/or overexpression of the receptor tyrosine kinase HER2 occurs in 20-25% of breast cancers, and is associated with poor prognosis. Targeting of HER2 with drugs such as trastuzumab, lapatinib, or pertuzumab has led to clinical benefit in patients with both metastatic and early-stage HER2-amplified breast cancer. However, resistance and disease progression always occurs in patients with metastatic disease, and many patients with early-stage breast cancer experience recurrences despite adjuvant anti-HER2 therapy. As such, understanding the mechanisms of resistance to anti-HER2 therapy has important clinical implications. Recent studies have identified mutations in PIK3CA, the gene encoding the catalytic subunit of Phosphatidylinositol 3 kinase (PI3K), as one mechanism of resistance to trastuzumab. However, such mutations are present in only a fraction of trastuzumab-resistant breast cancers. We therefore sought to uncover novel mechanisms of resistance to anti-HER2 therapy through an unbiased screen for kinases and kinase-related molecules that are able to rescue HER2-amplified breast cancer cells from HER2 inhibition. We utilized a library of nearly 600 lentivirally-delivered open reading frames (ORFs) to constitutively express the coding sequence of each molecule individually in HER2-amplified BT474 breast cancer cells in arrayed high-throughput format. We conducted two parallel screens for the ability of each of these molecules to rescue cells from anti-HER2 therapy: one in which we treated the cells with a lapatinib-like drug that inhibits the kinase activity of HER2 and EGFR, and one in which we lentivirally delivered a short hairpin RNA that suppresses expression of HER2. We identified those ORFs that restored viability of BT474 cells to greater than two standard deviations above the median of all ORFs in each screen. Multiple members of the MAPK and PI3K signaling pathways scored in both screens, serving to validate the approach. In addition, the survival kinases PIM1 and PRKACA scored robustly. Mechanistic studies suggest that these kinases may confer resistance by restoring the phosphorylation of, and thereby inactivating, the pro-apoptotic protein BAD. Consistent with this finding, overexpression of Bcl-xl, which is inhibited by BAD, also conferred resistance to lapatinib in HER2-amplified breast cancer cells. Furthermore, pharmacological blockade of Bcl-xl and Bcl-2 with ABT-263 enhanced lapatinib-induced killing of HER2-amplified breast cancer cells in vitro, and partially abrogated the rescue conferred by both PRKACA and PIM1. These findings suggest that combined inhibition of HER2 and the anti-apoptotic molecules Bcl-xl and Bcl-2 could enhance tumor cell eradication and prevent or delay the emergence of resistant disease. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-08-01.

Background: Dysregulated Wnt/β-catenin signaling has been linked to several cancers, including pancreatic cancer (PC) and colorectal cancer (CRC). WNT974 (formerly LGK974) - a first-in-class, selective, oral inhibitor of Porcupine (an O-acyltransferase required for Wnt activation and secretion) - has shown preclinical activity in tumor models with mutations upstream in the Wnt pathway. Furthermore, dysregulated Wnt signaling has been linked to T-cell exclusion in tumor tissue and resistance to immunotherapy, suggesting WNT974 may act synergistically with checkpoint inhibitors. Methods: This ongoing Phase I, open-label, dose-escalation and -expansion study (NCT01351103) is designed to determine the maximum tolerated dose and/or recommended dose for expansion, characterize the safety and tolerability, and assess preliminary antitumor activity, pharmacokinetic (PK), and pharmacodynamic properties of WNT974, alone or combined with PDR001 (spartalizumab, an anti-PD-1 antibody) in advanced/metastatic solid tumors. Here, we focus on preliminary biomarker analyses from the single-agent part of the study. The study initially enrolled pts with lobular breast cancer and melanoma and was later amended to enroll pts with triple negative breast cancer (TNBC), PC, and CRC, and those with tumors with genetic alterations upstream in the Wnt pathway. Pre and on-treatment skin and tumor tissue specimens were collected for RT-PCR analysis of AXIN2, a marker of Wnt pathway activity. NanoString gene expression was measured in a subset of pts using remnant RNA from the tumor samples, and chemokine and dendritic cell signatures were analyzed pre and on-treatment. Results: At the data cut-off date, March 2, 2017, 94 pts were enrolled. Median age was 58.5 years (range, 28-77), 43% were male, and the most common cancer types were PC (30%), melanoma (26%), and breast cancer (21%). Patients received single-agent WNT974 orally at doses of 5, 7.5, 10, 15, 20, 22.5, or 30 mg daily, 30 or 45 mg intermittently (4 days on, 3 days off), or 5 mg twice daily. Median duration of exposure was 4.9 weeks (range, 0.1-27.7). Safety, tolerability, and PK have previously been reported. AXIN2 expression in paired samples from skin and tumor showed evidence of Wnt pathway inhibition in all indications; this was not dose-dependent in the dose range studied. Immune signature analyses of a paired tumor sample subset (n=8) revealed an inverse association between change in AXIN2 expression and change in chemokine and activated dendritic cell signatures. Conclusions: Biomarker analyses show that WNT974 can potently inhibit Wnt pathway activity in skin and tumors. Immune signature data suggest that WNT974 treatment may promote T-cell recruitment into tumors, and support investigation of the combination of WNT974 with immunotherapy. The combination part of this study evaluating WNT974 combined with PDR001 (spartalizumab) is ongoing. Citation Format: Jordi Rodon, Guillem Argilés, Roisin M. Connolly, Ulka Vaishampayan, Maja de Jonge, Elena Garralda, Marios Giannakis, David C. Smith, Jason R. Dobson, Margaret McLaughlin, Abdelkader Seroutou, Yan Ji, Sinead Dolan, Jennifer Morawiak, Susan Moody, Filip Janku. Biomarker analyses from a phase I study of WNT974, a first-in-class Porcupine inhibitor, in patients (pts) with advanced solid tumors [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 CT175.

Most anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung tumors initially respond to small-molecule ALK inhibitors, but drug resistance often develops. After tumors develop resistance to highly potent 2nd-generation ALK inhibitors, approximately half harbor ALK resistance mutations, while the other half have other mechanisms of resistance. The latter often have activation of at least one of several different tyrosine kinases driving resistance. Such tumors are not expected to respond to the 3rd-generation ALK inhibitor, lorlatinib, which is able to overcome all clinically identified ALK resistance mutations, and further therapeutic options are limited. Herein, we deployed an shRNA screen of 1000 genes in multiple ALK inhibitor-resistant patient-derived cells (PDC) to discover sensitizers to ALK inhibition. This approach identified SHP2, a non-receptor protein tyrosine phosphatase, as a common targetable resistance node in multiple PDCs. SHP2 provides a parallel survival input downstream of multiple tyrosine kinases that promote resistance to ALK inhibitors. The recently discovered small-molecule SHP2 inhibitor, SHP099, in combination with the ALK TKI (tyrosine kinase inhibitor), ceritinib, halted the growth of resistant PDCs by preventing compensatory RAS and ERK1/2 reactivation. These findings suggest that combined ALK and SHP2 inhibition may be a promising therapeutic strategy for resistant cancers driven by several different ALK-independent resistance mechanisms. Citation Format: Leila Dardaei, Hui Qin Wang, Manrose Singh, Paul Fordjour, Satoshi Yoda, Grainne Kerr, Jinsheng Liang, Yichen Cao, Yan Chen, Justin Gainor, Luc Friboulet, Ibiayi Dagogo-Jack, David Myers, Emma Labrot, David Ruddy, Melissa Parks, Dana Lee, Richard DiCecca, Susan Moody, Huaixiang Hao, Morvarid Mohseni, Matthew LaMarche, Juliet Williams, Keith Hoffmaster, Giordano Caponigro, Alice Shaw, Aaron Hata, Cyril Benes, Fang Li, Jeffrey Engelman. SHP2 inhibition restores sensitivity to ALK inhibitors in resistant ALK-rearranged NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A145.

Despite development of highly potent and selective inhibitors (e.g., ceritinib, alectinib, lorlatinib) targeting anaplastic lymphoma kinase (ALK), resistance invariably develops and limits the efficacy of these inhibitors in the clinic. The major classes of resistance are on-target genetic alterations (e.g., secondary ALK kinase domain mutations) and activation of alternative or bypass signaling pathways. While most patients are responsive to sequential treatment with two or more ALK inhibitors, ALK-independent resistance eventually emerges and leads to failure of further ALK-directed monotherapy. We used a synthetic lethal pooled shRNA screen to discover loss-of-function events that could sensitize resistant patient-derived cell lines to ALK inhibition. In addition to identifying known bypass targets such as FGFR, EGFR and SRC, we also identified PTPN11 (which encodes SHP2, a non-receptor protein tyrosine phosphatase that modulates signaling downstream of growth factor receptors) as a common hit shared by cell lines exhibiting different mechanisms of bypass activation. In parallel with the shRNA screen, we also performed a high throughput combination compound screen in the same patient-derived models, and identified activation of the same bypass signaling pathways. We showed that the highly potent and selective small-molecule SHP2 inhibitor SHP099 could sensitize resistant cell lines to ALK inhibition. In biochemical studies, co-targeting of ALK and SHP2 overcame resistance mediated by ALK-independent bypass mechanisms by decreasing RAS-GTP loading potential of cells and inhibiting phospho-ERK rebound. These results suggest that dual ALK and SHP2 inhibition may represent a new therapeutic strategy for ALK-positive patients, whose lung cancers have evolved ALK-independent mechanisms of resistance, including activation of bypass signaling pathways. Citation Format: Leila Dardaei, Hui Qin Wang, Paul Fordjour, Manrose Singh, Grainne Kerr, Satoshi Yoda, Jinsheng Liang, Yichen Cao, Yan Chen, Justin F. Gainor, Luc Friboulet, Ibiayi Dagogo-Jack, David T. Myers, Emma Labrot, David Ruddy, Melissa Parks, Dana Lee, Richard H. DiCecca, Susan Moody, Huaixiang Hao, Morvarid Mohseni, Matthew LaMarche, Juliet Williams, Keith Hoffmaster, Giordano Caponigro, Cyril H. Benes, Alice T. Shaw, Aaron N. Hata, Fang Li, Jeffrey A. Engelman. SHP2 inhibition restores sensitivity to ALK inhibition in resistant ALK-rearranged non-small cell lung cancer (NSCLC) [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 1007. doi:10.1158/1538-7445.AM2017-1007