Somatic mutations of ERBB2 and ERBB3 (which encode HER2 and HER3, respectively) are found in a wide range of cancers. Preclinical modelling suggests that a subset of these mutations lead to ...constitutive HER2 activation, but most remain biologically uncharacterized. Here we define the biological and therapeutic importance of known oncogenic HER2 and HER3 mutations and variants of unknown biological importance by conducting a multi-histology, genomically selected, 'basket' trial using the pan-HER kinase inhibitor neratinib (SUMMIT; clinicaltrials.gov identifier NCT01953926). Efficacy in HER2-mutant cancers varied as a function of both tumour type and mutant allele to a degree not predicted by preclinical models, with the greatest activity seen in breast, cervical and biliary cancers and with tumours that contain kinase domain missense mutations. This study demonstrates how a molecularly driven clinical trial can be used to refine our biological understanding of both characterized and new genomic alterations with potential broad applicability for advancing the paradigm of genome-driven oncology.
The FDA approved irreversible inhibitor of ERBB1/2/4, neratinib, was recently shown to rapidly down-regulate the expression of ERBB1/2/4 as well as the levels of c-MET and mutant K-RAS via autophagic ...degradation. In the present studies, in a dose-dependent fashion, neratinib reduced the expression levels of mutant K-RAS or of mutant N-RAS, which was augmented in an additive to greater than additive fashion by the HDAC inhibitors sodium valproate and AR42. Neratinib could reduce PDGFRα levels in GBM cells, that was enhanced by sodium valproate. Knock down of Beclin1 or of ATG5 prevented neratinib and neratinib combined with sodium valproate / AR42 from reducing the expression of mutant N-RAS in established PDX and fresh PDX models of ovarian cancer and melanoma, respectively. Neratinib and the drug combinations caused the co-localization of mutant RAS proteins and ERBB2 with Beclin1 and cathepsin B. The drug combination activated the AMP-dependent protein kinase that was causal in enhancing HMG Co A reductase phosphorylation. Collectively, our data reinforce the concept that the irreversible ERBB1/2/4 inhibitor neratinib has the potential for use in the treatment of tumors expressing mutant RAS proteins.
Mutations in
, the gene encoding epidermal growth factor receptor (EGFR) family member HER2, are common in and drive the growth of "HER2-negative" (not
amplified) tumors but are rare in ..."HER2-positive" (
amplified) breast cancer. We analyzed DNA-sequencing data from HER2-positive patients and used cell lines and a patient-derived xenograft model to test the consequence of HER2 mutations on the efficacy of anti-HER2 agents such as trastuzumab, lapatinib, and neratinib, an irreversible pan-EGFR inhibitor. HER2 mutations were present in ~7% of HER2-positive tumors, all of which were metastatic but not all were previously treated. Compared to HER2 amplification alone, in both patients and cultured cell lines, the co-occurrence of HER2 mutation and amplification was associated with poor response to trastuzumab and lapatinib, the standard-of-care anti-HER2 agents. In mice, xenografts established from a patient whose HER2-positive tumor acquired a D769Y mutation in HER2 after progression on trastuzumab-based therapy were resistant to trastuzumab or lapatinib but were sensitive to neratinib. Clinical data revealed that six heavily pretreated patients with tumors bearing coincident HER2 amplification and mutation subsequently exhibited a statistically significant response to neratinib monotherapy. Thus, these findings indicate that coincident HER2 mutation reduces the efficacy of therapies commonly used to treat HER2-positive breast cancer, particularly in metastatic and previously HER2 inhibitor-treated patients, as well as potentially in patients scheduled for first-line treatment. Therefore, we propose that clinical studies testing the efficacy of neratinib are warranted selectively in breast cancer patients whose tumors carry both amplification and mutation of
/HER2.
We examined the role of
-activating mutations in endocrine therapy resistance in estrogen receptor positive (ER+) breast cancer.
mutation frequency was determined from large genomic databases. ...Isogenic knock-in
mutations in ER+ MCF7 cells and xenografts were used to investigate estrogen-independent growth. Structural analysis was used to determine the molecular interaction of
with HER3. Small molecules and siRNAs were used to inhibit PI3Kα, TORC1, and HER3.
Genomic data revealed a higher rate of
mutations in metastatic versus primary ER+ tumors. MCF7 cells with isogenically incorporated
kinase domain mutations exhibited resistance to estrogen deprivation and to fulvestrant both
and
, despite maintaining inhibition of ERα transcriptional activity. Addition of the irreversible HER2 tyrosine kinase inhibitor neratinib restored sensitivity to fulvestrant. HER2-mutant MCF7 cells expressed higher levels of p-HER3, p-AKT, and p-S6 than cells with wild-type HER2. Structural analysis of the HER2
variant implicated a more flexible active state, potentially allowing for enhanced dimerization with HER3. Treatment with a PI3Kα inhibitor, a TORC1 inhibitor or HER3 siRNA, but not a MEK inhibitor, restored sensitivity to fulvestrant and to estrogen deprivation. Inhibition of mutant HER2 or TORC1, when combined with fulvestrant, equipotently inhibited growth of MCF7/
xenografts, suggesting a role for TORC1 in antiestrogen resistance induced by
mutations.
mutations hyperactivate the HER3/PI3K/AKT/mTOR axis, leading to antiestrogen resistance in ER+ breast cancer. Dual blockade of the HER2 and ER pathways is required for the treatment of ER+/HER2 mutant breast cancers.
Summary Background The limitations of current treatments for postherpetic neuralgia (PHN) have led to the investigation of localised, non-systemic alternatives. NGX-4010, a high-concentration (8%) ...capsaicin dermal patch, was developed to treat patients with neuropathic pain. We report the results of a randomised, double blind, 12-week study of the efficacy and safety of one application of NGX-4010 in patients with PHN. Methods In this multicentre, double-blind, parallel-group trial, 402 patients were randomly assigned to one 60-min application of NGX-4010 (640 μg/cm2 8% capsaicin) or a low-concentration capsaicin control patch (3·2 μg/cm2 0·04% capsaicin). Patients were aged 18–90 years, had had postherpetic neuralgia for at least 6 months, and had an average baseline numeric pain rating scale (NPRS) score of 3 to 9. The primary efficacy endpoint was percentage change in NPRS score from baseline to weeks two to eight. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov , number NCT00115310. Findings Patients who were randomly assigned to NGX-4010 (n=206) had a significantly greater reduction in pain during weeks two to eight than did patients who had the control patch (n=196). The mean changes in NPRS score were −29·6% vs −19·9% (difference −9·7%, 95% CI −15·47 to −3·95; p=0·001). 87 (42%) patients who received NGX-4010 and 63 (32%) controls had a 30% or greater reduction in mean NPRS score (odds ratio OR 1·56, 95% CI 1·03 to 2·37; p=0·03). The patients who had NGX-4010 had significant improvements in pain during weeks two to 12 (mean change in NPRS score −29·9% vs −20·4%, difference −9·5, −15·39 to −3·61; p=0·002). Transient blood pressure changes associated with changes in pain level were recorded on the day of treatment, and short-lasting erythema and pain at the site of application were common, self-limited, and generally mild to moderate in the NGX-4010 group and less frequent and severe in the controls. Interpretation One 60-min application of NGX-4010 provided rapid and sustained pain relief in patients with postherpetic neuralgia. No adverse events were associated with treatment except for local reactions at the site of application and those related to treatment-associated pain. Funding NeurogesX.
Regorafenib is approved for the treatment of colorectal cancer and hepatocellular carcinoma. In the trial NCT02466802, we have discovered that regorafenib can be safely combined with the ...phosphodiesterase 5 inhibitor sildenafil in advanced solid tumor patients. The present studies determined whether the approved ERBB1/2/4 and RAS downregulating drug neratinib, could enhance the lethality of regorafenib + sildenafil. Neratinib enhanced regorafenib + sildenafil lethality in a greater than additive fashion in colon cancer cells. The drug combination reduced the expression of mutant K‐RAS and of multiple histone deacetylase (HDAC) proteins that required autophagosome formation. It caused green fluorescent protein or red fluorescent protein–tagged forms of K‐RAS V12 to localize into large intracellular vesicles. Compared with regorafenib + sildenafil, the three‐drug combination caused greater and more prolonged activation of the ATM‐AMPK‐ULK‐1 pathway and caused a greater suppression and prolonged inactivation of mammalian target of rapamycin, AKT, and p70 S6K. Approximately 70% of enhanced lethality caused by neratinib required ataxia‐telangiectasia‐mutated (ATM)–AMP‐dependent protein kinase (AMPK) signaling whereas knockdown of Beclin1, ATG5, FADD, and CD95 completely prevented the elevated killing effect. Exposure of cells to regorafenib + sildenafil reduced the expression of the checkpoint immunotherapy biomarkers programmed death‐ligand 1, ornithine decarboxylase, and indoleamine 2,3‐dioxygenase‐1 and increased the expression of major histocompatibility complex A (MHCA), which also required autophagosome formation. Knockdown of specific HDAC proteins recapitulated the effects observed using chemical agents. In vivo, using mouse cancer models, neratinib significantly enhanced the antitumor efficacy of regorafenib + sildenafil. Our data support performing a new three drug Phase I trial combining regorafenib, sildenafil, and neratinib.
Neratinib augments the lethality of regorafenib + sildenafil in vitro and in vivo. Neratinib is now fully validated as a drug that can cause the intracellular vesicularization and downregulation of mutant RAS proteins.
We developed neratinib-resistant HER2-mutant cancer cells by gradual dose escalation. RNA sequencing identified TORC1 signaling as an actionable mechanism of drug resistance. Primary and acquired ...neratinib resistance in HER2-mutant breast cancer patient-derived xenografts (PDXs) was also associated with TORC1 hyperactivity. Genetic suppression of RAPTOR or RHEB ablated P-S6 and restored sensitivity to the tyrosine kinase inhibitor. The combination of the TORC1 inhibitor everolimus and neratinib potently arrested the growth of neratinib-resistant xenografts and organoids established from neratinib-resistant PDXs. RNA and whole-exome sequencing revealed RAS-mediated TORC1 activation in a subset of neratinib-resistant models. DNA sequencing of HER2-mutant tumors clinically refractory to neratinib, as well as circulating tumor DNA profiling of patients who progressed on neratinib, showed enrichment of genomic alterations that converge to activate the mTOR pathway.
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•Neratinib resistance in HER2-mutant cancers is associated with TORC1 hyperactivation•Multiple mechanisms converge on TORC1 signaling to promote neratinib resistance•Combination with TORC1 inhibitor everolimus restores sensitivity to neratinib
In brief, Sudhan et al. identify that hyperactivation of TORC1 restores HER2 downstream signaling to drive neratinib resistance across histologically distinct HER2-mutant cancers. The combination of TORC1 inhibitor everolimus and neratinib arrests the growth of neratinib-resistant cells, organoids, and xenografts.
Endocrine therapies are the mainstay of treatment for oestrogen receptor (ER)-positive (ER
) breast cancer (BC). However, resistance remains problematic largely due to enhanced cross-talk between ER ...and growth factor pathways, circumventing the need for steroid hormones. Previously, we reported the anti-proliferative effect of everolimus (RAD001-mTORC1 inhibitor) with endocrine therapy in resistance models; however, potential routes of escape from treatment via ERBB2/3 signalling were observed. We hypothesised that combined targeting of three cellular nodes (ER, ERBB, and mTORC1) may provide enhanced long-term clinical utility.
A panel of ER
BC cell lines adapted to long-term oestrogen deprivation (LTED) and expressing ESR1
or ESR1
, modelling acquired resistance to an aromatase-inhibitor (AI), were treated in vitro with a combination of RAD001 and neratinib (pan-ERBB inhibitor) in the presence or absence of oestradiol (E2), tamoxifen (4-OHT), or fulvestrant (ICI182780). End points included proliferation, cell signalling, cell cycle, and effect on ER-mediated transactivation. An in-vivo model of AI resistance was treated with monotherapies and combinations to assess the efficacy in delaying tumour progression. RNA-seq analysis was performed to identify changes in global gene expression as a result of the indicated therapies.
Here, we show RAD001 and neratinib (pan-ERBB inhibitor) caused a concentration-dependent decrease in proliferation, irrespective of the ESR1 mutation status. The combination of either agent with endocrine therapy further reduced proliferation but the maximum effect was observed with a triple combination of RAD001, neratinib, and endocrine therapy. In the absence of oestrogen, RAD001 caused a reduction in ER-mediated transcription in the majority of the cell lines, which associated with a decrease in recruitment of ER to an oestrogen-response element on the TFF1 promoter. Contrastingly, neratinib increased both ER-mediated transactivation and ER recruitment, an effect reduced by the addition of RAD001. In-vivo analysis of an LTED model showed the triple combination of RAD001, neratinib, and fulvestrant was most effective at reducing tumour volume. Gene set enrichment analysis revealed that the addition of neratinib negated the epidermal growth factor (EGF)/EGF receptor feedback loops associated with RAD001.
Our data support the combination of therapies targeting ERBB2/3 and mTORC1 signalling, together with fulvestrant, in patients who relapse on endocrine therapy and retain a functional ER.
Purpose: Mutations associated with resistance to kinase inhibition are an important mechanism of intrinsic or acquired loss of clinical
efficacy for kinase-targeted therapeutics. We report the ...prospective discovery of ErbB2 mutations that confer resistance to
the small-molecule inhibitor lapatinib.
Experimental Design: We did in vitro screening using a randomly mutagenized ErbB2 expression library in Ba/F3 cells, which were dependent on ErbB2 activity for
survival and growth.
Results: Lapatinib resistance screens identified mutations at 16 different ErbB2 amino acid residues, with 12 mutated amino acids
mapping to the kinase domain. Mutations conferring the greatest lapatinib resistance cluster in the NH 2 -terminal kinase lobe and hinge region. Structural computer modeling studies suggest that lapatinib resistance is caused by
multiple mechanisms; including direct steric interference and restriction of conformational flexibility (the inactive state
required for lapatinib binding is energetically unfavorable). ErbB2 T798I imparts the strongest lapatinib resistance effect
and is analogous to the epidermal growth factor receptor T790M, ABL T315I, and cKIT T670I gatekeeper mutations that are associated
with clinical drug resistance. ErbB2 mutants associated with lapatinib resistance transformed NIH-3T3 cells, including L755S
and T733I mutations known to occur in human breast and gastric carcinomas, supporting a direct mechanism for lapatinib resistance
in ErbB2-driven human cancers. The epidermal growth factor receptor/ErbB2/vascular endothelial growth factor receptor inhibitor
EXEL-7647 was found to inhibit almost all lapatinib resistance-associated mutations. Furthermore, no ErbB2 mutations were
found to be associated with EXEL-7647 resistance and lapatinib sensitivity.
Conclusions: Taken together, these data suggest potential target-based mechanisms of resistance to lapatinib and suggest that EXEL-7647
may be able to circumvent these effects.
The irreversible ERBB1/2/4 inhibitor neratinib has been shown
to rapidly reduce the expression of ERBB1/2/4 and RAS proteins via autophagic/lysosomal degradation. We have recently demonstrated that ...neratinib and valproate interact to suppress the growth of 4T1 mammary tumors but had not defined whether the neratinib + valproate drug combination, in a mouse, had altered the biology of the 4T1 cells. Exposure of 4T1 mammary tumors to neratinib + valproate for three days resulted, two weeks later, in tumors that expressed less ERBB1, K-RAS, N-RAS, indoleamine-pyrrole 2,3-dioxygenase (IDO-1), ornithine decarboxylase (ODC) and had increased Class I MHCA expression. Tumors previously exposed to neratinib + valproate grew more slowly than those exposed to vehicle control and contained more CD8+ cells and activated NK cells. M1 but not M2 macrophage infiltration was significantly enhanced by the drug combination.
exposure of 4T1 tumor cells to neratinib + valproate variably reduced the expression of histone deacetylases 1-11.
, prior exposure of tumors to neratinib + valproate permanently reduced the expression of HDACs 1-3, 6 and 10. Combined knock down of HDACs 1/2/3 or of 3/10 rapidly reduced the expression IDO-1, and ODC and increased the expression of MHCA. H&E staining of normal tissues at animal nadir revealed no obvious cyto-architectural differences between control and drug-treated animals. We conclude that neratinib + valproate evolves 4T1 tumors to grow more slowly and to be more sensitive to checkpoint immunotherapy antibodies.
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