Combining Neratinib with CDK4/6, mTOR, and MEK Inhibitors in Models of HER2-positive Cancer

Abstract

Purpose: Neratinib is an irreversible, pan-HER tyrosine kinase inhibitor that is FDA approved for HER2-overexpressing/amplified (HER2 þ ) breast cancer. In this preclinical study, we explored the efficacy of neratinib in combination with inhibitors of downstream signaling in HER2 þ cancers in vitro and in vivo.Experimental Design: Cell viability, colony formation assays, and Western blotting were used to determine the effect of neratinib in vitro. In vivo efficacy was assessed with patient-derived xenografts (PDX): two breast, two colorectal, and one esophageal cancer (with HER2 mutations). Four PDXs were derived from patients who received previous HER2-targeted therapy. Proteomics were assessed through reverse phase protein arrays and network-level adaptive responses were assessed through Target Score algorithm.Results: In HER2 þ breast cancer cells, neratinib was synergistic with multiple agents, including mTOR inhibitors ever-olimus and sapanisertib, MEK inhibitor trametinib, CDK4/6 inhibitor palbociclib, and PI3Ka inhibitor alpelisib. We tested efficacy of neratinib with everolimus, trametinib, or palbociclib in five HER2 þ PDXs. Neratinib combined with everolimus or trametinib led to a 100% increase in median event-free survival (EFS; tumor doubling time) in 25% (1/4) and 60% (3/5) of models, respectively, while neratinib with palbociclib increased EFS in all five models. Network analysis of adaptive responses demonstrated upregulation of EGFR and HER2 signaling in response to CDK4/6, mTOR, and MEK inhibition, possibly providing an explanation for the observed synergies with neratinib.Conclusions: Taken together, our results provide strong preclinical evidence for combining neratinib with CDK4/6, mTOR, and MEK inhibitors for the treatment of HER2 þ cancer.

PI3K and MAPK Pathways as Targets for Combination with the Pan-HER Irreversible Inhibitor Neratinib in HER2-Positive Breast Cancer and TNBC by Kinome RNAi Screening

“…To the best of our knowledge, this present preclinical study is the first to show that neratinib can potentially induce TGI in both HER2+ breast cancer and TNBC when cotreated with inhibitors of the PI3K/Akt-mTOR or MAPK pathway. Our findings in HER2+ breast cancer were supported by a recent study by Zhao et al [27]. We also reported the novel finding of the potential anti-tumor effect of neratinib in both in vitro and in vivo xenograft models.…”

“…Intriguingly, the studies by others also demonstrated a significant antitumor effect when the PI3K inhibitor was combined with anti-HER2 therapy. Neratinib has a synergistic antitumor effect with everolimus in PIK3CA-mutated HER2+ breast cancer patient-derived xenograft models and with trametinib in HER2-mutated HER2+ breast cancer patient-derived xenograft models [27]. Combination treatment with lapatinib and ipatasertib overcomes resistance to anti-HER2 therapy in PIK3CA-mutant HER2+ breast cancer cells [29].…”

Cellular mechanisms underlying response and resistance to CDK4/6 inhibitors in the treatment of hormone receptor-positive breast cancer

“…Most studies searching for effective combination therapies that enhance CDK4/6i efficacy in ER-positive breast cancer have focused on concomitant inhibition of growth factor signaling pathways. Combined inhibition of CDK4/6 and growth factor receptors, such as HER2 and FGFR, or the downstream pathway members, such as PI3K, PDK1, and mTOR, has demonstrated synergy or at least heightened effects [36,[65][66][67][68][69]. In some cases, the effect of combined inhibition of CDK4/6 and a growth factor pathway is enhanced cytostasis or senescence [36,65,69], and in others, the effect is apoptosis [30,[66][67][68].…”

“…Combined inhibition of CDK4/6 and growth factor receptors, such as HER2 and FGFR, or the downstream pathway members, such as PI3K, PDK1, and mTOR, has demonstrated synergy or at least heightened effects [36,[65][66][67][68][69]. In some cases, the effect of combined inhibition of CDK4/6 and a growth factor pathway is enhanced cytostasis or senescence [36,65,69], and in others, the effect is apoptosis [30,[66][67][68]. The combination of ET, CDK4/6, and PI3K inhibition results in maximal growth inhibition in ER-positive breast cancer models [30].…”

“…Despite the range of effective combination regimens that has been explored preclinically, the molecular mechanisms underlying their additive or synergistic effects have not been clearly delineated. One common theme is a rebound increase in activity of upstream pathways (such as the PI3K pathway) in luminal breast cancer cells treated with CDK4/6i [ 30 , 47 , 66 – 68 ]. We have also reported that CDK4/6i increases the phosphorylation of HER family receptor tyrosine kinases and AKT in luminal HER2-positive cell lines [ 36 ].…”