Supplementary methods: PTEN screening

B: The dashed line represents 5 CTCs/7.5 mL. C: White circles indicate the location of the lymph node lesion with documented PR, demonstrating a 46% reduction in size from baseline. CT, computerized tomography; CTC, circulating tumor cells; PR, partial response; PSA, prostate-specific antigen.

Dashed lines represent 95% confidence intervals. PCWG2, Prostate Cancer Working Group 2; PSA, prostate-specific antigen.

Supplementary Table 1. PCWG2 criteria for progression.

Supplementary Figure 1: Study design

Supplementary Figure 4: Investigation of the p.L1049R mutation identified in a patient with castration-resistant prostate cancer.

Supplementary Table 7: Next-generation sequencing of tumor biopsy samples (see separate Excel file) Supplementary Table 8: Copy number variations for patients with tumor biopsy samples available (see separate Excel file) Supplementary Table 9: Summary of copy number variations as assessed by polymerase chain reaction (see separate Excel file)

LLQ=15.6 mg/mL. LLQ shown by horizontal reference line. N=1 for 400 mg GSK2636771 plus 160 mg enzalutamide 160 mg escalation cohort. LLQ, lower limit of quantification.

Abstract NRG1 rearrangements are oncogenic drivers that are enriched in invasive mucinous adenocarcinomas (IMA) of the lung. The oncoprotein binds ERBB3–ERBB2 heterodimers and activates downstream signaling, supporting a therapeutic paradigm of ERBB3/ERBB2 inhibition. As proof of concept, a durable response was achieved with anti-ERBB3 mAb therapy (GSK2849330) in an exceptional responder with an NRG1-rearranged IMA on a phase I trial (NCT01966445). In contrast, response was not achieved with anti-ERBB2 therapy (afatinib) in four patients with NRG1-rearranged IMA (including the index patient post-GSK2849330). Although in vitro data supported the use of either ERBB3 or ERBB2 inhibition, these clinical results were consistent with more profound antitumor activity and downstream signaling inhibition with anti-ERBB3 versus anti-ERBB2 therapy in an NRG1-rearranged patient-derived xenograft model. Analysis of 8,984 and 17,485 tumors in The Cancer Genome Atlas and MSK-IMPACT datasets, respectively, identified NRG1 rearrangements with novel fusion partners in multiple histologies, including breast, head and neck, renal, lung, ovarian, pancreatic, prostate, and uterine cancers. Significance: This series highlights the utility of ERBB3 inhibition as a novel treatment paradigm for NRG1-rearranged cancers. In addition, it provides preliminary evidence that ERBB3 inhibition may be more optimal than ERBB2 inhibition. The identification of NRG1 rearrangements across various solid tumors supports a basket trial approach to drug development. Cancer Discov; 8(6); 686–95. ©2018 AACR. See related commentary by Wilson and Politi, p. 676. This article is highlighted in the In This Issue feature, p. 663

Supplementary Figure 3: Glucose and insulin levels after treatment with GSK2636771 in fasted mice.

Supplementary Table 2. Summary of serious adverse events.

Supplementary Figure 2: LNCAP and DU-145 prostate cancer cells were treated with 1 or 10 μM GSK2636771 for up to 48 hours and probed with the indicated antibodies

Supplementary Table 1: 3 + 3 dose escalation guidelines Supplementary Table 2: Customized sequencing panel Supplementary Table 3: Summary of AEs occurring in >20% of all patients, treatment-related AEs and SAEs Supplementary Table 4: PK parameters following single dose oral dosing of GSK2636771 (PK population) Supplementary Table 5: PK parameters following single daily oral dosing of GSK2636771 (PK population) Supplementary Table 6: PK parameters following multiple dose oral dosing of GSK2636771 (PK population)

Figures S1, S2, S3, S4, and S5

Abstract Purpose: In patients with metastatic castration-resistant prostate cancer (mCRPC), resistance to androgen receptor (AR)-targeted therapies, such as enzalutamide, remains an issue. Inactivation of inhibitory PTEN activates PI3K/AKT signaling and contributes to resistance to androgen deprivation therapy and poor outcomes. Therefore, dual targeting of AR and PI3K/AKT pathways may limit tumor growth and reverse resistance. Patients and Methods: In this phase I study (NCT02215096), patients with PTEN-deficient mCRPC who progressed on prior enzalutamide received once-daily enzalutamide 160 mg plus PI3Kβ inhibitor GSK2636771 at 300 mg initial dose, with escalation or de-escalation in 100-mg increments, followed by dose expansion. Primary objectives were to evaluate safety/tolerability, determine the recommended phase II dose, and assess the 12-week non-progressive disease (PD) rate. Results: Overall, 37 patients were enrolled; 36 received ≥1 dose of GSK2636771 (200 mg: n = 22; 300 mg: n = 12; 400 mg: n = 2) plus 160 mg enzalutamide. Dose-limiting toxicities occurred in 5 patients (200 mg: n = 1; 300 mg: n = 2, 400 mg: n = 2). No new or unexpected adverse events or evidence of drug–drug interaction were observed. At the recommended dose of GSK2636771 (200 mg) plus enzalutamide, the 12-week non-PD rate was 50% (95% confidence interval: 28.2–71.8, n = 22); 1 (3%) patient achieved a radiographic partial response lasting 36 weeks. Four of 34 (12%) patients had prostate-specific antigen reduction of ≥50%. Conclusions: Although there was acceptable safety and tolerability with GSK2636771 plus enzalutamide in patients with PTEN-deficient mCRPC after failing enzalutamide, limited antitumor activity was observed.

Abstract Background GSK2849330, an anti‐HER3 monoclonal antibody that blocks HER3/Neuregulin 1 (NRG1) signaling in cancer cells, is engineered for enhanced antibody‐dependent cellular cytotoxicity and complement‐dependent cytotoxicity. This phase I, first‐in‐human, open‐label study assessed the safety, pharmacokinetics (PK), pharmacodynamics, and preliminary activity of GSK2849330 in patients with HER3‐expressing advanced solid tumors. Patients and Methods Patients with various tumor types were prospectively selected for HER3 expression by immunohistochemistry; a subset was also screened for NRG1 mRNA expression. In the dose‐escalation phase, patients received GSK2849330 1.4–30 mg/kg every 2 weeks, or 3 mg/kg or 30 mg/kg weekly, intravenously (IV). In the dose‐expansion phase, patients received 30 mg/kg GSK2849330 IV weekly. Results Twenty‐nine patients with HER3‐expressing cancers, of whom two expressed NRG1, received GSK2849330 (dose escalation: n = 18, dose expansion: n = 11). GSK2849330 was well tolerated. No dose‐limiting toxicities were observed. The highest dose, of 30 mg/kg weekly, expected to provide full target engagement, was selected for dose expansion. Treatment‐emergent adverse events (AEs) were mostly grade 1 or 2. The most common AEs were diarrhea (66%), fatigue (62%), and decreased appetite (31%). Dose‐proportional plasma exposures were achieved, with evidence of HER3 inhibition in paired tissue biopsies. Of 29 patients, only 1 confirmed partial response, lasting 19 months, was noted in a patient with CD74‐NRG1‐rearranged non‐small cell lung cancer (NSCLC). Conclusion GSK2849330 demonstrated a favorable safety profile, dose‐proportional PK, and evidence of target engagement, but limited antitumor activity in HER3‐expressing cancers. The exceptional response seen in a patient with CD74‐NRG1‐rearranged NSCLC suggests further exploration in NRG1‐fusion–positive cancers. Implications for Practice This first‐in‐human study confirms that GSK2849330 is well tolerated. Importantly, across a variety of HER3‐expressing advanced tumors, prospective selection by HER3/NRG1 expression alone was insufficient to identify patients who could benefit from treatment with this antibody‐dependent cell‐mediated cytotoxicity– and complement‐dependent cytotoxicity–enhanced anti‐HER3 antibody. The only confirmed durable response achieved was in a patient with CD74‐NRG1‐rearranged lung cancer. This highlights the potential utility of screening for NRG1 fusions prospectively across tumor types to enrich potential responders to anti‐HER3 agents in ongoing trials.

Introduction: NRG1 rearrangements produce chimeric ligands that subvert the ERBB pathway to drive tumorigenesis. A better understanding of the signaling networks that mediate transformation by NRG1 fusions is needed to inform effective therapeutic strategies. Unfortunately, this has been hampered by a paucity of patient-derived disease models that faithfully recapitulate this molecularly defined cancer subset. Methods: Patient-derived xenograft (PDX) and cell line models were established from NRG1-rearranged lung adenocarcinoma samples. Transcriptomic, proteomic, and biochemical analyses were performed to identify activated pathways. Efficacy studies were conducted to evaluate HER3- and MTOR-directed therapies. Results: We established a pair of PDX and cell line models of invasive mucinous lung adenocarcinoma (LUAD) (LUAD0061AS3, SLC3A2-NRG1), representing the first reported paired in vitro and in vivo model of NRG1-driven tumors. Growth of LUAD-0061AS3 models was reduced by the antiHER3 antibody GSK2849330. Transcriptomic profiling revealed activation of the MTOR pathway in lung tumor samples with NRG1 fusions. Phosphorylation of several MTOR effectors (S6 and 4EBP1) was higher in LUAD0061AS3 cells compared with human bronchial epithelial cells and the breast cancer cell line MDA-MB-175-VII (DOC4-NRG1 fusion). Accordingly, LUAD-0061AS3 cells were more sensitive to MTOR inhibitors than MDA-MB-175VII cells and targeting the MTOR pathway with rapamycin blocked growth of LUAD-0061AS3 PDX tumors in vivo. In contrast, MDA-MB-175-VII breast cancer cells had higher MAPK pathway activation and were more sensitive to MEK inhibition. Conclusions: We identify the MTOR pathway as a candidate vulnerability in NRG1 fusion-positive lung adenocarcinoma that may warrant further preclinical evaluation, with the eventual goal of finding additional therapeutic options for patients in whom ERBB-directed therapy fails. Moreover, our results uncover heterogeneity in downstream oncogenic signaling among NRG1-rearranged cancers, possibly tumor type-dependent, the therapeutic significance of which requires additional investigation. (c) 2021 Published by Elsevier Inc. on behalf of International Association for the Study of Lung Cancer.

NRG1 rearrangements are oncogenic drivers that are enriched in invasive mucinous adenocarcinomas (IMA) of the lung. The oncoprotein binds ERBB3-ERBB2 heterodimers and activates downstream signaling, supporting a therapeutic paradigm of ERBB3/ERBB2 inhibition. As proof of concept, a durable response was achieved with anti-ERBB3 mAb therapy (GSK2849330) in an exceptional responder with an NRG1-rearranged IMA on a phase I trial (NCT01966445). In contrast, response was not achieved with anti-ERBB2 therapy (afatinib) in four patients with NRG1-rearranged IMA (including the index patient post-GSK2849330). Although in vitro data supported the use of either ERBB3 or ERBB2 inhibition, these clinical results were consistent with more profound antitumor activity and downstream signaling inhibition with anti-ERBB3 versus anti-ERBB2 therapy in an NRG1-rearranged patient-derived xenograft model. Analysis of 8,984 and 17,485 tumors in The Cancer Genome Atlas and MSK-IMPACT datasets, respectively, identified NRG1 rearrangements with novel fusion partners in multiple histologies, including breast, head and neck, renal, lung, ovarian, pancreatic, prostate, and uterine cancers.Significance: This series highlights the utility of ERBB3 inhibition as a novel treatment paradigm for NRG1-rearranged cancers. In addition, it provides preliminary evidence that ERBB3 inhibition may be more optimal than ERBB2 inhibition. The identification of NRG1 rearrangements across various solid tumors supports a basket trial approach to drug development. Cancer Discov; 8(6); 686-95. ©2018 AACR.See related commentary by Wilson and Politi, p. 676This article is highlighted in the In This Issue feature, p. 663.

Elevated expression of the c-MYC oncogene (either due to gene amplification, translocation, abnormality in upstream signaling pathways and/or protein stabilization) is one of the most common abnormalities in human cancers. Efforts to identify direct pharmacological inhibitors of c-MYC function have not yet yielded drug-like molecules. Therefore, we sought to pursue alternative screening strategies for this classically “undruggable” transcription factor. We developed a novel antibody-based high-throughput HTRF screening assay that specifically detects endogenous c-MYC protein levels in a MYC amplified cancer cell line. Taking advantage of the short half-life of c-MYC, both at the protein and mRNA level, we conducted a cell-based screen of the GSK screening collection to identify compounds that can rapidly decrease c-MYC protein levels. Elimination of false positive hits using stringent triage assays successfully identified two valid hit series exemplified by GSK970 and GSK417. Molecular mode of action studies revealed these molecules inhibit MYC transcription by binding to the minor groove of DNA with AT sequence specificity. Unfortunately, this mechanism of c-MYC inhibition demonstrated poor in vivo translatability as tissue DNA acts as a molecular sink, effectively sequestering compound, and limiting its pharmacodnyamic response. Citation Format: Biju Mangatt, Anthony D. Pietra, Anna Waszkiewicz, Jon-Paul Jaworski, Sonja Ghidelli-Disse, Thomas J. Berrodin, Christian S. Sherk, Derrick W. Meinhold, Anna Rutkowska-Klute, Shanker K. Sundaram, Gopinath Ganji, Wendy S. Halsey, George P. Livi, William Li, James Mack, Stuart P. Romeril, Elisabeth A. Minthorn, Rakesh Kumar, Gerard C. Drewes, Dirk A. Heerding, Lorena A. Kallal, Carolyn A. Buser, Jesus R. Medina. Discovery of small-molecule compounds targeting c-MYC using a novel cell-based screen [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 2919.

Abstract Background: The PI3K/protein kinase B (AKT) pathway is commonly activated in several tumor types. Selective targeting of p110β could result in successful pathway inhibition while avoiding the on- and off-target effects of pan-PI3K inhibitors. GSK2636771 is a potent, orally bioavailable, adenosine triphosphate-competitive, selective inhibitor of PI3Kβ. Methods: We evaluated the safety, pharmacokinetics, pharmacodynamics and antitumor activity of GSK2636771 to define the recommended phase II dose (RP2D). During the dose-selection and dose-escalation stages (parts 1 and 2), patients with PTEN-deficient advanced solid tumors received escalating doses of GSK2636771 (25–500 mg once daily) using a modified 3+3 design to determine the RP2D; tumor type-specific expansion cohorts (part 3) were implemented to further assess tumor responses at the RP2D. Results: A total of 65 patients were enrolled; dose-limiting toxicities were hypophosphatemia and hypocalcemia. Adverse events included diarrhea (48%), nausea (40%), and vomiting (31%). Single- and repeat-dose exposure increased generally dose proportionally. GSK2636771 400 mg once daily was the RP2D. Phospho/total AKT ratio decreased with GSK2636771 in tumor and surrogate tissue. A castrate-resistant prostate cancer (CRPC) patient harboring PIK3CB amplification had a partial response for over a year; an additional 10 patients derived durable (≥24 weeks) clinical benefit, including two other patients with CRPC with PIK3CB alterations (≥34 weeks). GSK2636771 400 mg once daily orally induced sufficient exposure and target inhibition with a manageable safety profile. Conclusions: Genomic aberrations of PIK3CB may be associated with clinical benefit from GSK2636771. Clin Cancer Res; 23(19); 5981–92. ©2017 AACR.