Introduction:

Given acquired resistance patterns to androgen deprivation therapy and second-generation androgen receptor (AR) inhibitors, it is crucial to identify and target the mechanisms by which prostate cancer cells persist despite AR inhibition. Previous work has shown that the upregulation and secretion of stromal-derived growth factor neuregulin 1 (NRG1) activates PI3K-AKT signaling through its binding to the HER2/HER3 receptor and promotes prostate cancer cell growth during antiandrogen treatment. Here, we determine the clinical context where this tumor microenvironment derived NRG1 impacts response to AR inhibition. Additionally, we show that selective inhibition of NRG1 mediated activation of HER2/HER3 signaling with a clinical grade bispecific humanized immunoglobulin G1, zenocutuzumab (Zeno, MCLA-128), in both in vitro and in vivo models can overcome resistance to AR-targeted therapies.

Methods:

We used purified epithelial (22Pc-EP) and fibroblast (22Pc-CAF) cell lines derived the patient-derived xenograft model (CWR22Pc), which is initially castration sensitive but can progress to castration resistant. The 22Pc-EP line is endogenous PTEN wild-type (22Pc-EP^WT) and PTEN knock-out cells (22Pc-EP^PTEN) were generated using CRISPR-Cas9. Cells were stimulated with either recombinant NRG1 or conditioned media derived from the 22Pc-CAFs. Lysates were collected and prepared for western blot analysis to assess for activation of downstream signaling cascades, both with and without zenocutuzumab and enzalutamide treatment. Growth assays were also performed under similar conditions. 22Pc-EP^WT and 22Pc-EP^PTEN  cells were co-injected subcutaneously (10:1 ratio) in the flanks of castrated male mice then treated with either vehicle control, castration + enzalutamide (30mg/kg/day), or enzalutmide + zenocutuzumab (25mg/kg intraperitoneal injection weekly) once tumors were established. Tumor volume was measured bi-weekly and surviving mice were sacrificed after 7 weeks of therapy.

Results:

Stimulation with recombinant NRG1 was able to activate downstream PI3K signaling in the 22Pc-EP cell lines, and this signal was more robust and sustained over time in the setting of PTEN loss. 22Pc-EP^WT cells were responsive to enzalutamide and cell growth was decreased upon treatment, though this was overcome by stimulation with NRG. Treatment with zenocutuzumab restored the enzalutamide sensitive phenotype. 22Pc-EP^PTEN cells were also sensitive to enzalutamide, but NRG1 stimulation paradoxically decreased cell growth and no response to zenocutuzumab was observed (Fig 1). Additionally, culturing cells with the 22Pc-CAF conditioned media conferred analogous results to recombinant NRG1 stimulation. In our in vivo experiment, enzalutamide significantly decreased tumor growth in both the PTEN wild-type and knock-out setting. However, only PTEN wild-type tumors were responsive to zenocutuzumab (Fig 2).

Conclusion:

Here, we show that NRG1 promotes prostate cancer cell survival and resistance to AR inhibition in PTEN wild-type prostate cancer cells. Activation of downstream PI3K-AKT signaling in a prostate cancer epithelial cell line was seen after both recombinant NRG1 stimulation and culturing with cancer associated fibroblast conditioned media. This supports a paracrine mediated action of NRG1 whereby it is secreted by stromal cells in the tumor microenvironment to promote resistance through PI3K signaling. Using zenocutuzumab, a novel clinical grade inhibitor of the NRG1-HER2/3 interaction, we were able to restore an androgen sensitive phenotype in both in vitro and in vivo models in the PTEN wild-type setting. High-risk PTEN wild-type prostate cancers may therefore be an appropriate population for future clinical studies.

Funding: This work was funded by a Conquer Cancer – GlaxoSmithKline Oncology Young Investigator Award. Any opinions, findings, and conclusions expressed in this material are those of the author(s) and do not necessarily reflect those of the American Society of Clinical Oncology® or Conquer Cancer®