The irreversible ERBB1/2/4 inhibitor neratinib interacts with the BCL-2 inhibitor venetoclax to kill mammary cancer cells

ABSTRACT

The irreversible ERBB1/2/4 inhibitor, neratinib, down-regulates the expression of ERBB1/2/4 as well as the levels of MCL-1 and BCL-XL. Venetoclax (ABT199) is a BCL-2 inhibitor. At physiologic concentrations neratinib interacted in a synergistic fashion with venetoclax to kill HER2 + and TNBC mammary carcinoma cells. This was associated with the drug-combination: reducing the expression and phosphorylation of ERBB1/2/3; in an eIF2α-dependent fashion reducing the expression of MCL-1 and BCL-XL and increasing the expression of Beclin1 and ATG5; and increasing the activity of the ATM-AMPKα-ULK1 S317 pathway which was causal in the formation of toxic autophagosomes. Although knock down of BAX or BAK reduced drug combination lethality, knock down of BAX and BAK did not prevent the drug combination from increasing autophagosome and autolysosome formation. Knock down of ATM, AMPKα, Beclin1 or over-expression of activated mTOR prevented the induction of autophagy and in parallel suppressed tumor cell killing. Knock down of ATM, AMPKα, Beclin1 or cathepsin B prevented the drug-induced activation of BAX and BAK whereas knock down of BID was only partially inhibitory. A 3-day transient exposure of established estrogen-independent HER2 + BT474 mammary tumors to neratinib or venetoclax did not significantly alter tumor growth whereas exposure to [neratinib + venetoclax] caused a significant 7-day suppression of growth by day 19. The drug combination neither altered animal body mass nor behavior. We conclude that venetoclax enhances neratinib lethality by facilitating toxic BH3 domain protein activation via autophagy which enhances the efficacy of neratinib to promote greater levels of cell killing.

KEYWORDS:

• Autophagy
• receptor tyrosine kinase
• neratinib
• venetoclax
• ABT199

Introduction

Over-expression of the epidermal growth factor receptor (EGFR, HER1, ERBB1) is recognized as a biomarker for tumor cell growth, invasion and resistance to chemotherapy [1, and references therein]. ERBB1 is frequently expressed in triple negative breast cancer (TNBC). Other members of this receptor family, ERBB2, ERBB3 and ERBB4, have also been linked to the oncogenic drug-resistant phenotype.^2-4 The recently FDA approved HER2+ mammary carcinoma therapeutic, the ERBB1/2/4 inhibitor neratinib, was previously shown by our group to rapidly down-regulate the expression of ERBB1/2/3/4, c-MET, PDGFRα and mutant K-/N-RAS proteins.^5,6 When we microscopically examined neratinib-treated cells we observed the appearance of large and small vesicles inside the cell, close to the plasma membrane and within the cytoplasm that contained ERBB family receptors and the mutant RAS proteins, and which co-stained for Beclin1, phosphorylated ATG13 S318 and/or cathepsin B and LAMP2. Other investigators have made similar observations for neratinib, examining ERBB2 and cathepsin B expression.⁷ The precise mechanisms by which neratinib rapidly causes receptor tyrosine kinase internalization and degradation are unknown.

Venetoclax (ABT199) is a BCL-2 specific inhibitor FDA approved for use in a subtype of chronic lymphocytic leukemia; CLL cells with a 17p deletion are survival-addicted to the elevated expression of BCL-2.^8,9 The drug acts to facilitate the dissociation of toxic BH3 domain proteins, e.g. BAX and BAK, from BCL-2, thereby permitting the toxic BH3 domain proteins to form pores in the mitochondrial outer membrane thereby causing the release into the cytosol of cytochrome c and AIF.^10,11 The release of AIF and cytochrome c activates effector caspases and necroptotic killing mechanisms.¹² We have recently demonstrated that neratinib can induce an endoplasmic reticulum stress response in tumor cells and that this response is causal in the drug reducing the protein levels of MCL-1 and BCL-XL.^5,6 We reasoned that if neratinib was reducing MCL-1 and BCL-XL expression, and ABT199 was inhibiting BCL-2, we may be able to observe a synergy of anti-tumor effects when the drugs were combined. i.e. the simultaneous release of multiple toxic BH3 domain proteins.

The present studies demonstrate that neratinib and ABT199 interact in a synergistic fashion to kill mammary carcinoma cells (ER+, HER2+ and TNBC). The primary mechanisms of cell killing involved DNA damage signaling and receptor down-regulation reducing the activity of mTOR which resulted in the formation of toxic autophagosomes. Autophagosome formation and the autolysosome protease cathepsin B played essential roles upstream of the mitochondrion in causing BAX and BAK activation, and down-stream of the mitochondrion AIF- and caspase 9- dependent tumor cell killing.

Results

The safe achievable plasma C_max value for neratinib is ∼150 nM and for the BCL-2 inhibitor venetoclax (ABT199) is ∼1.0 μM. Neratinib and ABT199 at clinically relevant concentrations interacted in an additive to greater than additive fashion to kill mammary carcinoma cells in short-term cell viability assays (Fig. 1A and B). In colony formation assays designed to assess synergy via median dose effect analyses, using BT474 HER2+ and SUM149 TNBC cells, neratinib and ABT199 synergized to cause clonogenic cell death with combination index values of less than 1.0 (Fig. 1C and D). We have recently published that neratinib, in addition to inhibiting ERBB family receptor tyrosine kinase activity, could also cause the internalization and auto-lysosome-mediate degradation of these receptors, and of mutant RAS proteins. Treatment of BT474 and SUM149 cells with [neratinib + ABT199] reduced both the phosphorylation and expression of ERBB1, ERBB2 and ERBB3 (Fig. 1E). In both cell lines, the protein level of ERBB2 was reduced to a significantly greater extent than the reduction in ERBB2 phosphorylation. In pancreatic tumor cells expressing a mutant K-RAS and ovarian cancer cells expressing a mutant N-RAS, [neratinib + ABT199] reduced the expression of ERBB1 / ERBB2 and K-/N-RAS proteins to a similar extent, and with a similar reduction to that observed in BT474 mammary carcinoma cells (Fig. 1F).

Figure 1. Neratinib and ABT199 combine to kill breast cancer cells. A. Breast cancer cells were treated with vehicle control, neratinib (50 nM), ABT199 (0.25 μM) or the drugs in combination for 24 h. Cell viability was determined by trypan blue exclusion assay (n = 3 +/− SEM) # p < 0.05 greater than ABT199 alone. B. Breast cancer cells were treated with vehicle control, neratinib (50 nM), ABT199 (0.25 μM) or the drugs in combination for 24 h. Cell viability was determined by live / dead assay (n = 3 +/− SEM). # p < 0.05 greater than ABT199 alone. C. BT474 cells and D. SUM149 cells were plated in 6-well plates in sextuplicate as individual cells (500 cells per well). After 12 h the cells were treated with vehicle control, neratinib, ABT199 or the drugs combined, at the indicated concentrations in the figure, at a fixed ratio. After 24 h, the media is removed, the cells washed with warm drug-free media, and fresh drug-free media placed on the cells. After 10 days, colonies of > 50 cells have formed and the cells are fixed in place and stained with crystal violet. The plating efficiency under each treatment condition is determined and the fraction affected determined. Synergy was determined using the Calcusyn for Windows program using the method of Cho and Talalay (n = 2 independent studies in sextuplicate). A combination index of less than 1.00 indicates a synergy of drug interaction. E. BT474 and SUM149 cells were treated with vehicle control or with [neratinib (50 nM) + ABT199 (250 nM)] for 6 h. Cells were fixed in place and immunostaining performed to detect the total expression and phosphorylation of ERBB1, ERBB2 and ERBB3 (n = 120 cells +/− SEM) # p < 0.05 lower level of protein expression compared to protein phosphorylation. F. Spiky (ovarian), PANC1 (pancreatic) and BT474 (mammary) were treated with vehicle control or with [neratinib (50 nM) + ABT199 (250 nM)] for 6 h. Cells were fixed in place and immunostaining performed to detect the total expression of ERBB1, ERBB2, N-RAS, K-RAS and ERK2 (n = 120 cells +/− SEM) # p < 0.05 lower level of protein expression compared to vehicle control.

We next performed a series of broad agnostic screening studies examining well-recognized pathway activities downstream of ERBB receptors and RAS, after [neratinib + ABT199] exposure, as well as those involved in sensing DNA damage and autophagy regulation. After 6 h of exposure, the combination of [neratinib + ABT199] reduced the phosphorylation of ERK1/2, AKT T308, mTOR S2441 and ULK-1 S757 (Fig. 2A). At the same time point the drug combination increased the phosphorylation of eIF2α S51, ATM S1981, γH2AX, AMPKα T172, Raptor S792, TSC2 T1462, ULK-1 S317 and ATG13 S318. The phosphorylation of NFκB S536 was not altered by the drug combination in either cell line tested. This data argues that we are inducing an endoplasmic reticulum stress response, a DNA damage response, and likely inducing autophagosome formation. In both cell lines [neratinib + ABT199] treatment reduced BAD S112 phosphorylation, implying its activation as a toxic BH3 domain protein, and increased the protein levels of Beclin1, BIM, ATG5, LAMP2 and p62 (Fig. 2B). The total expression levels of BAX and BAK were not altered. These findings further suggest that the drug combination is inducing autophagy. The drug combination reduced expression of the mitochondrial protective proteins MCL-1 and BCL-XL (Fig. 2C). Knock down of eIF2α prevented the drug combination from decreasing BCL-XL and MCL-1 expression and increasing Beclin1 and ATG5 expression (Fig. 2D).

Figure 2. Neratinib and ABT199 interact to activate ATM-AMPK-ULK1 signaling and eIF2α signaling that correlates with reduced expression of MCL-1 and BCL-XL. A. SUM149 and BT474 cells were treated with vehicle control or [neratinib (50 nM) + niraparib (2.0 μM)] for 6 h. The cells were fixed in place and immunostaining performed to determine the phosphorylation of the indicated proteins at 10X magnification (data from multiple separate images & treatments +/− SEM) #p < 0.05 less than vehicle control; #p < 0.05 greater than vehicle control. B. SUM149 and BT474 cells were treated with vehicle control or [neratinib (50 nM) + niraparib (2.0 μM)] for 6 h. The cells were fixed in place and immunostaining performed to determine the expression of the indicated proteins at 10X magnification (data from multiple separate images & treatments +/− SEM) #p < 0.05 less than vehicle control; #p < 0.05 greater than vehicle control. C. SUM149 and BT474 cells were treated with vehicle control or [neratinib (50 nM) + niraparib (2.0 μM)] for 6 h. The cells were fixed in place and immunostaining performed to determine the expression of MCL-1 and BCL-XL (n = 120 cells +/− SEM) #p < 0.05 less staining intensity than vehicle control.

We then performed another series of broad agnostic screening studies examining the molecular mechanisms by which [neratinib + ABT199] killed breast cancer cells. In BT474 cells over-expression of the caspase 8/10 inhibitor c-FLIP-s, the mitochondrial protective protein BCL-XL or dominant negative caspase 9 significantly reduced drug combination -induced killing whereas in SUM149 cells over-expression of c-FLIP-s was not protective (Fig. 3A and B). However, in both cell lines, neither knock down of the death receptor CD95 nor the adaptor protein FADD significant reduced cell killing, suggesting that in BT474 cells caspase 8/10 activity in a FADD -independent fashion was facilitating tumor cell death. Knock down of toxic BH3 domain protein expression; BAD, BAK, BAX, or BIM, significantly reduced killing as did knock down of eIF2α, or knock down of the ATM-AMPK-ULK1-Beclin1/ATG5-cathepsin B pathway. Thus, the observed activation of eIF2α plays a positive role in cell killing as does activation of the DNA damage response pathway as it leads into the regulation of autophagosome formation and to autolysosome proteases.

Figure 3. Neratinib and ABT199 kill via toxic autophagy and necroptotic processes. A. and B. SUM149 and BT474 cells were transfected with: a scrambled siRNA molecules or siRNA molecules to knock down the expression of CD95, AIF, AMPKα, ATG5, ATM, BAD, BAX, BAK, Beclin1, BIM, cathepsin B, eIF2α, FADD and ULK-1. In parallel other portions of SUM149 and BT474 cells were transfected with an empty vector plasmid (CMV) or with plasmids to express c-FLIP-s, BCL-XL or dominant negative caspase 9. Twenty-four h after transfection cells were treated with vehicle control or with [neratinib (50 nM) + ABT199 (0.25 μM)] in combination for 24 h. Cell viability was determined by live / dead assay (n = 3 +/− SEM). #p < 0.05 less than vehicle control; ##p < 0.01 less than vehicle control; ###p < 0.005 less than vehicle control. C. SUM149 and BT474 cells were transfected with an empty vector plasmid (CMV) or with plasmids to express activated mTOR or activated MEK1. Twenty-four h after transfection cells were treated with vehicle control or with [neratinib (50 nM) + ABT199 (0.25 μM)] in combination for 24 h. Cell viability was determined by live / dead assay (n = 3 +/− SEM). #p < 0.05 less than corresponding value in CMV transfected. D. SUM149 and BT474 cells were transfected with an empty vector plasmid (CMV) or with a plasmid to express activated MEK1. Twenty-four h after transfection cells were treated with vehicle control or with [neratinib (50 nM) + ABT199 (0.25 μM)] in combination for 6 h. The expression of BIM was determined (n = 120 +/− SEM). # p < 0.05 less than corresponding value in CMV transfected cells.

It is well known that high levels of ERK1/2 activity can impede tumor cell killing by many drugs, and can in parallel suppress the ubiquitination and degradation of the toxic BH3 domain protein BIM.^13,14 Expression of activated mTOR, that suppresses the induction of autophagosome formation by [neratinib + ABT199], or activated MEK1, that suppresses the drug-induced increase in BIM expression, both significantly reduced [neratinib + ABT199] lethality (Fig. 3C and D).

We next examined the molecular mechanisms by which autophagosome and autolysosome levels were increased following [neratinib + ABT199] exposure. The drug combination, in an additive fashion, initially increased autophagosome formation that was temporally followed by an increase in autolysosome levels and a decrease in autophagosome levels (Fig. 4A and B). Knock down of [BAX+BAK], [BIM+BAD] or BID did not significantly alter the levels of drug-induced autophagosomes and autolysosomes whereas knock down of ATM, AMPKα or Beclin1, or over-expression of activated mTOR, significantly reduced autophagosome and autolysosome formation. Protective BH3 domain proteins such as BCL-2, BCL-XL and MCL-1 can act to suppress autophagosome formation by sequestering Beclin1.¹⁵ Over-expression of BCL-XL reduced the ability of ABT199 and [neratinib + ABT199] to increase autophagosome levels and reduced the ability of [neratinib + ABT199] to elevate autolysosome numbers (Fig. 4C and D). Similar data were obtained in SUM149 cells (Figures S1 and S2). Collectively, our findings argue that the primary driver of mTOR inactivation and autophagosome formation, neratinib, can induce autophagosomes in a manner independent of sequestered Beclin1 whereas the ability of ABT199 to cause autophagosome formation is partially blocked by BCL-XL that will sequester BH3 domain proteins such as BAX, BAK and Beclin1.

Figure 4. Increased levels of autophagosomes and autolysosomes require ATM-AMPK-ULK-1 signaling and not the actions of BAX/BAK, BIM/BAD or BID. A. and B. BT474 cells were transfected with a scrambled siRNA molecules or siRNA molecules to knock down the expression of ATM, AMPKα; Beclin1, BID, [BAX+BAK], [BIM+BAD] and with a plasmid to express LC3-GFP-RFP. Parallel portions of SUM149 and BT474 cells were transfected with an empty vector plasmid (CMV) or with a plasmid to express activated mTOR, and with a plasmid to express LC3-GFP-RFP. Twenty-four h after transfection cells were treated with vehicle control or [neratinib (50 nM) + ABT199 (0.25 μM)] in combination for 4 h and 8 h. The cells were imaged at 60X magnification and the mean number of intense fluorescent GFP+ and RFP+ foci in the cells determined (from 40 cells per condition in triplicate +/− SEM). #p < 0.05 less than corresponding value in siSCR/CMV cells; ##p < 0.01 less than corresponding value in siSCR/CMV cells. C. and D. BT474 cells were transfected with an empty vector plasmid or a plasmid to express BCL-XL, and with a plasmid to express LC3-GFP-RFP. Twenty-four h after transfection cells were treated with vehicle control or [neratinib (50 nM) + ABT199 (0.25 μM)] in combination for 4 h and 8 h. The cells were imaged at 60X magnification and the mean number of intense fluorescent GFP+ and RFP+ foci in the cells determined (from 40 cells per condition in triplicate +/− SEM). # p < 0.05 less than corresponding value in siSCR/CMV cells.

We then performed studies to define the drug-induced sequence and members of the DNA damage signaling pathway regulating autophagosome formation, and whether BCL-XL over-expression caused similar changes in their protein phosphorylation. The drug-induced phosphorylation of ATM S1981 was partially reduced by knock down of AMPKα or of eIF2α (Fig. 5A). Knock down of ATM almost abolished the drug-induced increase in AMPKα T172 phosphorylation whereas knock down of eIF2α had no effect. Knock down of ATM-AMPK signaling reduced the drug-induced reduction in ERK1/2, mTOR S2448 and of ULK-1 S757 phosphorylation and blocked the enhancement of ULK-1 S317 phosphorylation. Thus: DNA damage signaling from ATM-AMPK lowers mTOR / ULK-1 S757 and elevates ULK-1 S317 phosphorylation to achieve autophagosome formation. The same pathway acts to lower ERK1/2 phosphorylation, which we have previously linked to increased expression of the toxic BH3 domain protein BIM. However, the data also argue for a role of eIF2α in some of these signaling processes as knock down of eIF2α facilitates [neratinib + ABT199] to increase the phosphorylation of ERK1/2, AKT T308 and p70 S6K T389, and, the ability of the drug combination to enhance ULK-1 S317 phosphorylation in the absence of eIF2α is reduced.

Figure 5. Neratinib and ABT199 activate an ATM-AMPK pathway and cause ATG13 S318 phosphorylation. A. BT474 cells were transfected with a scrambled siRNA control or with siRNA molecules to knock down the expression of eIF2α, AMPKα or ATM. Twenty-four h after transfection, cells were treated with vehicle control or [neratinib (50 nM) + ABT199 (0.25 μM)] for 6 h. The cells were fixed in place and immunostaining performed to determine the expression and phosphorylation of the indicated proteins at 10X magnification. (data from multiple separate images & treatments +/− SEM) #p < 0.05 less than siSCR value; #p < 0.05 greater than siSCR value above vehicle treated baseline value; #p < 0.05 greater than siSCR value. B. BT474 cells were transfected with an empty vector plasmid (CMV) or with a plasmid to express BCL-XL. Twenty-four h after transfection, cells were treated with vehicle control or [neratinib (50 nM) + ABT199 (0.25 μM)] for 6 h. The cells were fixed in place and immunostaining performed to determine the expression and phosphorylation of the indicated proteins at 10X magnification. (data from multiple separate images & treatments +/− SEM) #p < 0.05 less than CMV value; ## p < less than baseline vehicle treated value; #p < 0.05 greater than CMV value above baseline.

Over-expression of BCL-XL partially reduced the drug-induced phosphorylation of ATM S1981, AMPKα T172, ULK-1 S317 and ATG13 S318 and abolished the drug-induced dephosphorylation of ERK1/2, AKT T308 and p70 S6K T389 (Fig. 5B). BCL-XL over-expression did not prevent drug-exposure from reducing mTOR S2448 or ULK-1 S757 phosphorylation. Similar data were obtained in SUM149 triple negative breast cancer cells (Figure S3). The data over-expressing BCL-XL argues that in the [neratinib + ABT199] system, mTOR-dependent regulation of ULK-1 S757 phosphorylation plays a greater role in controlling the process leading to toxic autophagosome production than does phosphorylation of ULK-1 S317.

As noted previously, by sequestration of Beclin1, proteins such as BCL-2, BCL-XL and MCL-1 have the potential to prevent autophagosome formation. Activation of toxic BH3 domain proteins, however, can displace Beclin1 from being sequestered permitting Beclin1 to promote autophagy. Previously we have shown that knock down of BAX, BAK, BIM, BID and BAD did not alter [neratinib + ABT199] -induced autophagy arguing that activation / over-expression of toxic BH3 domain proteins was not a primary mechanism for drug-induced cell death. Hence, we now asked the opposite question, is the activation of BAX and BAK by [neratinib + ABT199] dependent on prior autophagy or DNA damage signaling? Knock down of ATM, AMPKα, Beclin1 or [BIM + BAD] considerably reduced the ability of [neratinib + ABT199] to activate BAX (Fig. 6A). Knock down of BID, a protein that we had a priori assumed would be important for BAX activation as it is a substrate for both caspase 8 and cathepsin B, was much less effective at preventing BAX activation. However, knock down of BID, or ATM, AMPKα, Beclin1 or [BIM + BAD] prevented BAK activation. These data suggest that the DNA damage response and autophagy are upstream of BAX and BAK activation, and that the autolysosome protein cathepsin B plays an essential role upstream of the mitochondrion/caspase 9/AIF in mediating mitochondrial dysfunction and cell killing. Similar data were obtained in SUM149 cells (Figure S4).

Figure 6. [Neratinib + ABT199] -induced activation of BAX and BAK requires ATM-AMPK signaling. A. BT474 cells were transfected with a scrambled siRNA control or with siRNA molecules to knock down the expression of ATM, AMPKα, Beclin1, cathepsin B, [BIM+BAD] or BID. Twenty-four h after transfection, cells were treated with vehicle control or with [neratinib (50 nM) + ABT199 (0.25 μM)] for 6 h. Cells were lysed in CHAPS buffer to maintain the conformational status of BAX and BAK, and using activity specific antibodies, BAX and BAK were immunoprecipitated. BAX and BAK precipitates were subjected to SDS PAGE and western immunoblotting performed to determine the amount of activated BAX and BAK precipitated and from pre-precipitate lysate, the total protein levels of BAX and BAK. The percentage reduction in BAX / BAK staining was determined using Odyssey Imager intensity staining software (n = 3 +/− SEM) # p < 0.05 less than corresponding value in siSCR cells. B. BT474 cells (3 × 10⁶) were injected into the fourth mammary fat pad of female athymic mice and permitted to form tumors for four days and the mice segregated into four groups with ∼30 mm³ mean volumes. Mice were treated for three days with vehicle control (PBS and cremophore), neratinib (15 mg/kg QD), ABT199 (50 mg/kg QD), or the drugs in combination. Tumor volumes were determined on the days indicated in the graph up to day 19 (n = 10 / group +/− SEM). ## p < 0.05 less than neratinib.

Finally, we tested whether neratinib and ABT199 interacted in a model tumor system to suppress tumor growth. Established BT474 tumors were treated for 3 days with vehicle control, neratinib, ABT199 or the drugs combined. Neither neratinib alone nor ABT199 alone reduced tumor growth by day 19 (Fig. 6B). Tumors exposed to [neratinib + ABT199] for three days had a significantly slower re-growth than either tumor treated with a single agent. By day 19 the tumor growth delay comparing vehicle control to [neratinib + venetoclax] was 7 days (TC = 2.3). No alterations in animal body mass or behavior were observed following any drug exposure. These findings indicate that [neratinib + ABT199] at clinically relevant doses has putative translatability into a model living system.

Discussion

The present studies sought to prove or refute whether the novel approved ERBB1/2/4 inhibitor neratinib could interact with the approved BCL-2 inhibitor venetoclax to kill mammary carcinoma cells. In short term death assays the drugs combined in an additive to greater than additive fashion to kill ER+, HER2+ and TNBC mammary carcinoma cells. In long-term colony formation assays a synergy of tumor cell killing was observed combining neratinib with ABT199.

The reasoning behind the present studies was that we had previously observed neratinib reducing MCL-1 and BCL-XL expression and believed that the simultaneous inhibition / down-regulation of MCL-1, BCL-XL, and via venetoclax also BCL-2, would result in enhanced tumor cell killing. The combination of [neratinib + ABT199] strongly activated BAX and BAK. However, despite venetoclax theoretically being able to cause BAX and BAK activation as a single agent, by simply displacing BAX/BAK from BCL-2, strong activation of both toxic BH3 domain proteins required signaling via ATM-AMPK, the induction of autophagosomes and the actions of the autolysosome protease cathepsin B. In prior studies, we have linked the autophagic pro-death actions of cathepsin B on the ability of this protease to cause cleavage of BID.^16,17 BID is a target of caspase 8 and of cathepsin B, both acting to cause BID cleavage and its activation. Knock down of BID modestly reduced the activation of BAX but this reduced activation was still significantly greater than the amount of BAX activation with the expression of [BIM + BAD] knocked down. Knock down of BIM or BAD partially protected cells from [neratinib + ABT199] toxicity. This implies the neratinib-dependent inactivation of ERK1/2, and the subsequent dephosphorylation of BAD S112 and increased expression of BIM, play a greater role in BAX activation than does BID (Figure S5).

We also attempted to define the role of toxic BH3 domain protein sequestration, including sequestration of Beclin1, by over-expressing BCL-XL (n.b. whose function is not inhibited by venetoclax). BCL-XL over-expression partially reduced the induction of autophagosome formation but neither fully blocked autophagosome or autolysosome formation. Over-expression of BCL-XL did not prevent the drug-induced dephosphorylation of mTOR S2448 and the dephosphorylation of ULK-1 S757. BCL-XL over-expression did, however, reduce the activation of the ATM-AMPK-ULK-1 S317 / ATG13 S318 pathway and largely prevent the inactivation of ERK1/2, AKT and p70 S6K. Collectively these data argue that the primary pathway being modulated by [neratinib + ABT199] exposure links mTOR S2448, ULK-1 S757 and ATG13 S318. DNA damage signaling and inactivation of ERK1/2 are secondary events following this initial signal. The mechanisms by which autophagy can lead to DNA damage in our drug system will require studies beyond the scope of the present manuscript.

At present neratinib is approved as a neo-adjuvant treatment in HER2+ breast cancer for patients who have already completed trastuzumab therapy. Our most recent published studies have demonstrated that in addition to being an inhibitor of ERBB1/2/4 neratinib has the unexpected property of causing receptor internalization and subsequent degradation.^5,6 We then extended this observation to demonstrate that mutated RAS proteins associated in quaternary signaling complexes are also degraded in response to neratinib. These unexpected properties of neratinib can also help explain how and why the drug causes an endoplasmic reticulum stress response in tumor cells. Neratinib causes the degradation of HDAC6, the HDAC responsible for regulating HSP90 function. Reduced HDAC6 expression correlates with enhanced HSP90 acetylation and reduced HSP90 chaperone functionality. This will consequently result in greater levels of unfolded / denatured proteins in the cytosol which will lead to an ER stress response and eIF2α phosphorylation. Upon eIF2α activation the levels of proteins with relatively short half-lives, e.g. MCL-1 and BCL-XL, rapidly decline, as we have observed using neratinib. Future studies, based on our recent discovery combining neratinib with HDAC inhibitors, together with our data using ABT199 in the present manuscript point towards combining these three agents. Preliminary studies indicate that ABT199 profoundly enhances the lethality of [neratinib + valproate] in breast cancer cells (Dent & Booth, Unpublished Observations).¹⁸

At present, it is unclear whether Abbvie, the owners of venetoclax, will be interested in future translational studies exploring the interaction between their drug and neratinib in solid tumor patients. Our in vitro data has defined the mechanisms by which the drugs interact, and our in vivo data support the concept that the two drugs can interact in vivo to at least double the tumor control rate, without any apparent normal tissue toxicities in the mouse. In conclusion, our data strongly argue that the BCL-2 inhibitor venetoclax (ABT199), presently approved for the treatment of blood cancers, has potential anti-cancer utility in breast cancer when combined with the novel FDA approved ERBB1/2/4 inhibitor neratinib.

Materials and Methods

Materials. Venetoclax (ABT199) was purchased from Selleckchem (Houston, TX). Neratinib was supplied by Puma Biotechnology Inc. (Los Angeles, CA). Trypsin-EDTA, DMEM, RPMI, penicillin-streptomycin were purchased from GIBCOBRL (GIBCOBRL Life Technologies, Grand Island, NY). BT474, BT549 and MCF7 cells were purchased from the ATCC and were not further validated beyond that claimed by ATCC. Commercially available validated short hairpin RNA molecules to knock down RNA / protein levels were from Qiagen (Valencia, CA) (Figure S6, see below). Reagents / performance of experimental procedures were described in refs: 5, 6, 12 and 18. Antibodies used in the manuscript were: (a) from Cell Signaling Technology: AIF (5318), BAX (5023), BAK (12105), BAD (9239), BIM (2933), BAK1 (12105), Beclin1 (3495), cathepsin B (31718), CD95 (8023), FADD (2782), eIF2α (5324), P-eIF2α S51 (3398), ULK-1 (8054), P-ULK-1 S757 (14202), P-AMPK S51 (2535), AMPKα (2532), P-ATM S1981 (13050), ATM (2873), and ATG5 (12994). TSC2 (4308), P-TSC2 T1462 (3617), Raptor (2280), P-Raptor S792 (2083), mTOR (2983), P-mTOR S2448 (5536), P-mTOR S2481 (2974), ATG13 (13468), MCL-1 (94296), BCL-XL (2764), P-AKT T308 (13038), P-ERK1/2 (5726), P-STAT3 Y705 (9145), P-p65 S536 (3033), p62 (23214), LAMP2 (49067); (b) from Abgent: P-ULK-1 S317 (3803a); (c) from Novus Biologicals: P-ATG13 S318 (19127). The pre-validated smart-pool siRNA molecules used in the manuscript were from Qiagen: siSCR (SI03650318), ATM (SI00604737), cathepsin B (1027416), BAX (GS581), BAK (GS578); AMPKα (GS5562), BIM (GS10018), BAD (GS572), Beclin1 (GS8678), ATG5 (GS9474), CD95 (GS355), AIF (GS9131), eIF2α (GS83939), FADD (GS8772), ULK-1 (GS8408), ATG13 (GS9776).

Methods

Culture and in vitro drug treatments. All cell lines were cultured at 37°C (5% (v/v CO₂) in vitro using RPMI supplemented with 5% (v/v) fetal calf serum and 1% (v/v) Non-essential amino acids. For short term cell killing assays and immuno-staining studies, cells were plated at a density of 3 × 10³ per cm² and 24 h after plating treated with various drugs, as indicated. In vitro drug treatments were from a 100 mM stock solution of each drug and the maximal concentration of Vehicle carrier (VEH; DMSO) in media was 0.02% (v/v). Cells were not cultured in reduced serum media during any study. The safe achievable plasma C_max for neratinib is ∼150 nM; for venetoclax (ABT199) it is ∼1.0 μM.

Transfection of cells with siRNA or with plasmids

For Plasmids: Cells were transfected 24 h after plating. Plasmids expressing a specific mRNA or appropriate vector control plasmid DNA (CMV, control) were diluted in medium and in parallel Lipofectamine 2000 (Invitrogen), was diluted into medium. The samples were combined and incubated at room temperature for 30 min. This mixture was then added to each well of cells with incubation for an additional four hours. An equal volume of 2x medium was then added to each well. Cells were incubated for 24 h to permit protein expression, then treated with drugs.

Transfection for siRNA: Cells were transfected 24 h after plating. For transfection, an annealed negative control (a “scrambled” sequence with no significant homology to any known gene sequences from mouse, rat or human cell lines, siSCR) were used. Scrambled or experimental siRNA was diluted into media and combined with Hiperfect (Qiagen), according to manufacturer's instructions. This mixture was added to cells and added onto cells. Fresh media was added after 2 h followed by a further 24 h incubation before drug treatments.

Colony formation / isobologram assays. Single cells were plated in 6-well plates in sextuplicate as individual cells (500 cells per well). After 12 h the cells were treated for 24 h with vehicle control, neratinib, ABT199 or the drugs combined, at the indicated concentrations in the figure, at a fixed ratio. After 24 h the media is replaced with drug-free media. After ∼10 days, colonies of have formed. The cells are fixed, stained with crystal violet and the numbers of colonies counted. Synergy was determined using the Calcusyn for Windows program using the method of Cho and Talalay (n = 2 independent studies in sextuplicate). A combination index of less than 1.00 indicates a synergy of drug interaction. The Fraction affected data is plotted alongside the Combination Index.

Detection of cell viability, protein expression and protein phosphorylation by immuno-fluorescence using a Hermes WiScan wide-field microscope. http://www.idea-bio.com/, Cells (4 × 10³) are plated into a 96 well plate, genetically manipulated, and then 24 h later treated with drugs. For live/dead assays cells are isolated by centrifugation / cyto-spin to associate dead cells with the base of each well. Cells are treated with live/dead reagent and are visualized in the Hermes at 10X magnification. Green cells = viable; yellow/red cells = dying/dead. The numbers of viable and dead cells were counted manually from three images taken from each well combined with data from another two wells of separately treated cells. For immuno-fluorescence studies, cells are fixed in place and permeabilized and then pre-blocked with rat serum for 3 h. Cells are then incubated with a primary antibody to detect the expression / phosphorylation of a protein (usually at 1:100 dilution) overnight. Cells are washed, and incubated for 3 h with a secondary antibody added that has either a fluorescent red, green or blue chemical tag. The cells are visualized at either 10X to determine gross total fluorescence intensity staining for an antigen using software integral to the Hermes microscope or at 60X for fine detail and co-localization assessments in the Hermes microscope. The software automatically assesses and removes background fluorescence from each data set so that comparisons are made examining actual cell staining intensity between conditions / treatments.

Detection of cell death by Trypan Blue assay: Cells were treated with vehicle control or with neratinib / ABT199, alone or in combination. At the indicated time points cells were harvested by trypsinization and centrifugation. Cell pellets were resuspended in PBS and mixed with trypan blue agent. Viability was determined microscopically using a hemocytometer. Five hundred cells from randomly chosen fields were counted and the number of dead cells was counted and expressed as a percentage of the total number of cells counted.

Assessment of autophagy: Cells were transfected with a plasmid to express a green fluorescent protein (GFP) and red fluorescent protein (RFP) tagged form of LC3 (ATG8). Twenty-four h after transfection, cells were exposed to vehicle control or to drugs for 4 h and 8 h. The levels of GFP+ and RFP+ vesicles in the cells at each time point were examined at 60X magnification in the Hermes microscope system.

Animal studies: Studies were performed per USDA regulations under VCU IACUC protocol AD20008. BT474 cells (3 × 10⁶) were implanted into the 4^th mammary fat pad of athymic mice. Tumors were permitted to form (∼25-30 mm³) and animals segregated into groups with similar mean volumes. Animals were treated for three days via oral gavage with: vehicle control (cremophore); neratinib 15 mg/kg (QD Days 1, 2, 3); venetoclax 50 mg/kg (ABT199) (QD Days 1, 2, 3) or the drugs in combination. Tumor volumes were measured prior to drug administration and every three days after the initiation of therapeutic interventions up to Day 19. (n = 10 mice per group +/−SEM). When the volume of the tumor reached >2,000 mm,³ animals were humanely sacrificed.

Data analysis. Comparison of the effects of various treatments (performed in triplicate three times) was using one-way analysis of variance (ANOVA) and a two tailed Student's t-test. Statistical examination of in vivo animal survival data utilized both a two tailed Student's t-test and log rank statistical analyses between the different treatment groups. Differences with a p-value of < 0.05 were considered statistically significant. Experiments shown are the means of multiple individual points from multiple experiments (± SEM).

There are no conflicts of interest to report.

Abbreviations

ERK	=	extracellular regulated kinase
PI3K	=	phosphatidyl inositol 3 kinase
ca	=	constitutively active
dn	=	dominant negative
ER	=	endoplasmic reticulum
mTOR	=	mammalian target of rapamycin
MAPK	=	mitogen activated protein kinase
PTEN	=	phosphatase and tensin homologue on chromosome ten
ROS	=	reactive oxygen species
CMV	=	empty vector plasmid or virus
si	=	small interfering
SCR	=	scrambled
VEH	=	vehicle
HDAC	=	histone deacetylase

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

Support for the present study was funded from philanthropic funding from Massey Cancer Center, the Universal Inc. Chair in Signal Transduction Research and PHS R01-CA192613. Thanks to Dr. H.F. Young and the Betts family fund for support in the purchase of the Hermes Wiscan instrument. The authors have no conflicts of interest to report.

Additional information

Funding

HHS | NIH | National Cancer Institute (NCI), CA192614.