Rethinking Gamma-Secretase Inhibitors for Treatment of Non-Small Cell Lung Cancer: Is Notch the Target?

Author Manuscript Published OnlineFirst on August 13, 2018; DOI: 10.1158/1078-0432.CCR-18-1635 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Rethinking gamma-secretase inhibitors for treatment of non-small cell lung cancer: Is

Notch the target?

Sharon R. Pine, Ph.D.

Rutgers Cancer Institute of New Jersey, Rutgers, The State University of New Jersey, New

Brunswick, NJ, USA

Department of Pharmacology and Department of Medicine, Robert Wood Johnson Medical

School, Rutgers, The State University of New Jersey, New Brunswick, NJ, USA

Running title: Rethinking gamma secretase inhibitors

Keywords: gamma secretase inhibitor, Notch, patient-derived xenograft, non-small cell lung cancer, targets

Corresponding Author: Sharon R. Pine, Ph.D., Rutgers Cancer Institute of New Jersey,

Rutgers, The State University of New Jersey, New Brunswick, NJ, 08901. Phone (732) 235

9629; Fax (732) 235 8096; E-mail: [email protected]

Support: This research was supported by the National Cancer Institute (NCI), National Institutes of Health (NIH) (R01CA190578 to SRP).

Conflict of interest statement: None to disclose

Word count: Translational Relevance, 116; Abstract, 217; Manuscript, 2,522

Number of figures: 1 figure

Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 13, 2018; DOI: 10.1158/1078-0432.CCR-18-1635 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Translational Relevance

Non-small cell lung cancer is responsible for one-fourth of cancer deaths. Although targeted therapy is available for a subset of patients and immunotherapy has recently become a standard- of-care, the death toll due to NSCLC has not decreased much. We discuss the need for additional therapies, focusing on ɣ-secretase inhibitors, a class of small molecule compounds that are most commonly known for inhibiting the Notch pathway. We propose that Notch pathway mutations may not always be the intended target for ɣ-secretase inhibitors and furthermore, that other targets may serve as better biomarkers. Diligent identification and validation of the biomarkers that confer sensitivity to ɣ-secretase inhibitors are needed in order to continue clinical trials on molecularly selected patients.

Abstract

Lung cancer is the leading cause of cancer deaths among men and women. Gamma-secretase inhibitors, a class of small molecule compounds that target the Notch pathway, have been tested to treat non-small cell lung cancer (NSCLC) in pre-clinical and clinical trials. Although ɣ- secretase inhibitors elicit a response in some tumors as single agents and sensitize NSCLC to cytotoxic and targeted therapies, they have not yet been approved for NSCLC therapy. We discuss our recently published pre-clinical study using the ɣ-secretase inhibitor AL101, formerly

BMS906024, on cell lines and PDX models of NSCLC, primarily lung adenocarcinoma. We propose that Notch pathway mutations may not be the most suitable biomarker for predicting

NSCLC response to ɣ-secretase inhibitors. Gamma-secretases have over 100 known γ‐secretase cleavage substrates. Many of the ɣ-secretase substrates are directly involved in carcinogenesis or tumor progression, and are ideal candidates to be the “on-target” biomarkers for ɣ-secretase inhibitors. We propose the need to systematically test the ɣ-secretase and other targets as potential biomarkers for sensitivity before continuing clinical trials. Now that we have entered the post-genome/transcriptome era, this goal is easily attainable. Discovery of the biomarker(s) that predict sensitivity to ɣ-secretase inhibitors would guide selection of the responder population that is most likely to benefit and move the compounds closer to approval for therapeutic use in

NSCLC.

Current treatment of non-small cell lung cancer

The American Cancer Society has estimated that there will be 234,030 new cases of lung cancer and 154,050 deaths due to lung cancer in the United States in 2018, which ranks as the highest cause of deaths among all cancers for men and women [1]. Non-small cell lung cancer (NSCLC) accounts for 80 – 85% of patients with lung cancer. In early stages of the disease, patients are eligible for surgery that may be curative. Approximately 57% of NSCLC are metastatic [2], and until recently, these patients typically received platinum-based doublet chemotherapy or other two-drug combinations with only a 5% 5-year survival rate [3]. Recent breakthroughs in the use of immune checkpoint inhibitors in combination with chemotherapy or the use of targeted therapies have changed how advanced lung cancer patients are treated. Several immune checkpoint inhibitors have gone through clinical trials. The US Food and Drug Administration approved the use of pembrolizimab, an anti-PD1 inhibitor, for frontline treatment of all patients with metastatic non-squamous NSCLC who lack actionable mutations [4]. Based on the results of the phase III KEYNOTE-407 trial (NCT02775435), pembrolizumab may be approved for frontline treatment of metastatic lung squamous cell carcinoma soon. The addition of pembrolizumab to standard chemotherapy in unselected advanced-stage non-squamous NSCLC patients increased the median progression-free survival from 9 to 13 months [5]. The objective response rate for the group receiving pembrolizumab in addition to chemotherapy increased from about 30% in the chemotherapy-only group to 55%. Although these improvements are impressive when compared to historical advances, they highlight the fact that not all patients will benefit from the therapy. Frontline targeted therapies are available for NSCLC patients with a

BRAF V600E mutation, or patients with features of lung adenocarcinoma (LUAD) harboring actionable driver mutations in EGFR, or fusions involving ALK or ROS1. Unfortunately, the

nearly universal development of drug resistance has limited the success of targeted therapy [6].

This underscores the need to further refine the biomarkers for predicting the efficacy of immune checkpoint inhibitors and targeted therapies, and efforts are underway to address this on many fronts. The current state of NSCLC treatment overall highlights the urgent need to consider other strategies and to continue identifying impactful agents.

ɣ-secretase and ɣ-secretase inhibitors

Gamma secretase is a fascinating, multisubunit protein complex. Its catalytic core sits in the intramembrane space and facilitates the cleavage of single-pass transmembrane receptors, usually triggered by external signals. The ɣ-secretase complex comprises a catalytic subunit, called presenilin (PSEN1 and PSEN2), that works in concert with three other proteins, anterior pharynx defective 1 (APH1), presenilin enhancer 2 (PEN2), and Nicastrin [7, 8] (Fig. 1). Over

100 γ‐secretase substrates have been identified to date, including the four well-characterized mammalian Notch receptors (Notch1‐4) and the five canonical transmembrane Notch ligands [7].

Notch signaling, which has been reviewed elsewhere [7], is an evolutionarily conserved pathway that is crucial for the development and homeostasis of most tissues, and that has been implicated in cancer. The most commonly known substrate of gamma secretase is amyloid precursor protein

(APP) that, when cleaved by ɣ- and β-secretase, produces a short peptide called β-amyloid. The abnormally folded fibrillar form of β-amyloid is the primary component of amyloid plaques found in the brains of Alzheimer's disease patients [9]. Gamma secretase also cleaves many type-

I transmembrane proteins that have a known functional role in cancer, such as E-Cadherin,

CD44, DCC (deleted in colorectal cancer), IGF1R (insulin-like growth factor receptor), VEGF-

R1 (vascular endothelial growth factor receptor), the receptor tyrosine kinase ErbB4 (a.k.a.,

HER4), as well as numerous cytokine receptors involved in tumor progression including interleukin-6 receptor (IL-6R) and IL-1R [10].

Gamma secretase inhibitors (GSI) are a class of small-molecule inhibitors that, as the name implies, prevent the cleavage of γ‐secretase substrates. A number of GSIs were developed after

γ‐secretase was identified as an enzyme responsible for the accumulation of β-amyloid in

Alzheimer's disease [11, 12]. The various GSIs were comprehensively reviewed [13] and thus, are not discussed in this commentary. Despite the high expectations, GSIs were unsuccessful for treatment of Alzheimer’s disease. The first GSI to enter Phase III clinical trials for Alzheimer’s disease was not only ineffective, it accelerated cognitive decline, even though the GSI decreased

β-amyloid levels in a dose-dependent manner [14]. The reasons for the unsuccessful trials were initially believed to be caused by an infrequent dosing schedule and short half-life of the GSI, causing a re-bound effect in which γ‐secretase activity soared after the GSI was metabolized.

However, it was later proposed that deficient γ‐secretase, rather than increased γ‐secretase activity may be the contributor to Alzheimer’s disease [15]. Alternatively, some of the deficits in cognition may be related to Notch deficiency, because Notch is expressed in the neocortex, the hippocampus and the cerebellum, and is required for adult neurological function [16-18] .

Despite the disappointing outcome in Alzheimer’s disease, γ‐secretase was projected to have high potential as an effective therapeutic target in cancer [19, 20], primarily because it is involved in the activation of Notch signaling.

Efficacy of ɣ-secretase inhibitors for treatment of non-small cell lung cancer

We recently combined GSIs with chemotherapy to test if inhibiting Notch could sensitize

NSCLC, primarily lung adenocarcinoma, to standard cytotoxic treatments [21]. Although somatic mutations in the Notch pathway are rare in NSCLC [22-24], our rationale originated from numerous lines of evidence that Notch drives lung tumorigenesis, progression and cancer stem cell characteristics [25] [7, 20, 26-30]. For example, aberrant Notch activation can be a

“driver” of lung cancer development. When activated Notch1 intracellular domain (N1ICD) is conditionally overexpressed in the murine lung epithelium, it causes pulmonary adenomas, and when combined with conditional Myc overexpression, N1ICD significantly hastens Myc-induced development of lung adenocarcinoma [25]. Notch1 is also required for tumor initiation in KRAS- induced lung adenocarcinoma [26, 27]. A meta-analysis encompassing over 3,500 patients from

19 studies revealed that there is a significant correlation between overexpression of Notch1 and

Notch3 receptors, as well as the Notch ligands DLL3 and HES1, and poor outcome in NSCLC patients [28]. We previously reported that Notch activation in lung adenocarcinoma enhances the epithelial-mesenchymal phenotype, a known driver of tumor progression [29]. Notch inhibition sensitizes cancers to cytotoxic chemotherapy as well as targeted agents including anti-estrogens and HER2 inhibitors [7, 26, 31, 32]. Notch activation is known to participate in resistance to other targeted therapies such as anti-angiogenesis agents [33]. Thus, inhibiting Notch activation by GSIs could be beneficial for treatment of NSCLC.

The GSI AL101 (BMS906024) sensitizes NSCLC to chemotherapy

AL101, formerly called BMS906024 and recently acquired by Ayala Pharmaceuticals, is a potent

GSI with high bioavailability [34]. GSIs can have differing potencies for each of the 4 Notch paralogs, but AL101 was reported to inhibit processing of all 4 Notch receptors to a similar

degree (IC50 values of 1.6, 0.7, 3.4, and 2.9 nM for Notch1, -2, -3, and -4 receptors in cell-free assays) [34]. We administered AL101 in combination with front-line cytotoxic chemotherapy in preclinical models of NSCLC [35]. Using a set of 31 NSCLC cell lines, primarily lung adenocarcinoma, we observed that AL101 synergized with paclitaxel and cisplatin, though a much higher degree of synergy was observed with paclitaxel. In vivo studies in immunocompromised mice using NSCLC patient-derived xenografts (PDXs) confirmed the enhanced antitumor activity for AL101 plus paclitaxel versus either drug alone, through decreased cell proliferation and increased apoptosis [35]. It is important to note that none of the samples included in our study harbored activating mutations in Notch or inactivating mutations in Numb or FBXW7, the common negative regulators of activated Notch. Thus, as predicted, the efficacy of AL101 or other GSIs might extend beyond the rare subset of NSCLC patients whose tumors harbor activating Notch pathway mutations.

The synergy between AL101 and paclitaxel was significantly more potent, on average, in

NSCLC samples that were wild-type (WT) for KRAS and BRAF than in tumors harboring gain- of-function mutations in either KRAS or BRAF, both in cell culture and in pre-clinical mouse xenograft models. Moreover, when we knocked down the mutant KRAS allele in selected

NSCLC samples originally lacking synergy between AL101 and paclitaxel, synergy was significantly increased [35]. These findings suggested that, at least in some cases, constitutive

KRAS activation diminishes the effects of GSI-induced sensitivity to chemotherapy. The reasons for this are currently unclear. RAS oncogenes increase Notch1 expression and activity to maintain the RAS-transformed phenotype [36], and, as mentioned above, Notch signaling is required for KRAS-driven lung adenocarcinoma [26, 27]. Thus, one would expect that KRAS-

driven tumors would be at least partly dependent on Notch and sensitive to Notch inhibition. The data was consistent with a recent report that Notch inhibition alone has insufficient anti-tumor activity in KRAS-mutant PDXs [37]. It is possible that activated Notch participates in KRAS- induced carcinogenesis but is no longer required for maintenance of the tumorigenic state in established KRAS-mutant tumors.

Among the KRAS- and BRAF-mutant group of NSCLC cell lines treated with paclitaxel and

AL101 in our study, there was a large range of combination index (CI) values, with about half of the samples showing some level of synergy (conservatively defined as a CI value ≤ 0.7), and two samples showing a high degree of synergy (CI ≤ 0.2). The findings were likely due to the high inter-tumor heterogeneity due to varying secondary mutations known to be present within this subgroup [38, 39]. A comprehensive assessment of the genomic landscapes of the KRAS- or

BRAF-mutant tumors that were exquisitely sensitive to AL101 plus paclitaxel may help identify the responder population that would benefit from the combination therapy. This is particularly important because KRAS mutations are one of the most frequent oncogenic events in lung adenocarcinoma, are associated with poor prognosis, and are not yet “clinically targetable” [23,

40].

To further tease out the mechanisms driving GSI-induced sensitivity to paclitaxel in the KRAS- or BRAF-mutant subgroup, we determined that there was a significant correlation between synergy and mutant or null TP53 status [35]. These data were in contrast with clinical studies reporting that overexpression of activated Notch is associated with poor survival only in patients whose tumors express WT TP53 [41], and with previous findings that Notch1 controls tumor cell

survival via p53 [27]. The data imply that Notch1 may not have been the major target of the GSI, or that Notch inhibitors combined with paclitaxel can drive p53-independent cell death.

Biomarkers for selection of patients for GSI-based therapies

Notch1 is known to be activated by genotoxic chemotherapy across numerous cancer types [7,

42]. We also observed an increase in levels of activated Notch1 after paclitaxel treatment in a portion of the NSCLC samples. However, we were perplexed that the synergy between paclitaxel and AL101 did not correlate with chemotherapy-induced Notch1 activation in the NSCLC samples. It was also surprising that there was no significant correlation between the degree of synergy and basal levels of activated Notch1 or Notch3 in that same panel. Additionally, when we exogenously overexpressed the activated forms of Notch1 or Notch3, we were unable to reverse the enhanced sensitivity of NSCLC to paclitaxel by AL101. This led us to conclude that, at least in a subset of NSCLC, Notch may not be the primary target of AL101 that elicits the high degree of sensitivity to genotoxic chemotherapy. It is also possible that Notch inhibition and subsequent rescue is more complex, e.g., non-canonical pathways, and needs further examination.

The gold standard within the GSI field has been to demonstrate 1) the inhibitor hit the intended target, and 2) the compound had anti-tumor activity [34, 43-46]. However, what was generally lacking from preclinical GSI studies was ensuring that a rescue of the target could abrogate the anti-tumor activity. In some tumor types, there is irrefutable evidence that Notch is the target of

GSIs (e.g., T-cell acute lymphoblastic leukemia, triple-negative breast cancer or metastatic adenoid cystic carcinoma with Notch1 gain-of-function mutations) [47-49]. We propose that

high N1ICD expression may not be a suitable biomarker for selection of lung cancer patients to receive GSI therapy. Moreover, we propose that the other cancer types lacking prevalent Notch pathway mutations should be explored for biomarkers of GSI sensitivity.

As reviewed by Haapasalo & Kovacs, there are well over 100 known γ‐secretase cleavage substrates and possibly several hundred additional unknown targets [10]. Given the large number of substrates for γ-secretase, it would not be surprising if other cancer-related “on-target” effects of GSIs were responsible for GSI-induced sensitivity to paclitaxel. One potential target is the hyaluronate receptor CD44 (Fig. 1), a cancer stem cell marker that promotes tumor invasion and contributes to chemo-resistance of NSCLC [50]. Once cleaved by γ-secretase, CD44ICD translocates to the nucleus and acts as a transcriptional activator of tumor progression-related genes including Notch1, MMP9 and PDK1 [51, 52]. Further support for CD44 as a GSI target in cancer therapy is provided by reports that GSIs target cancer stem cells, as measured by a decrease in CD44 expression [53]. E-cadherin is another GSI target that might contribute to the anti-tumor activity of AL101 (Fig. 1). E-cadherin participates in maintenance of the epithelial phenotype [54]. Cleavage of E-cadherin by γ-secretase stimulates disassembly of the E‐ cadherin–β‐catenin complex, increases the cytosolic pool of β‐catenin, a key regulator of the Wnt signaling pathway, and promotes tumor cell survival as well as tumor progression [54]. Gamma- secretase also negatively regulates VEGF-R1. Treatment with GSIs causes rapid and abnormal blood vessel growth characterized by vessel occlusion, disrupted blood flow, and increased vascular leakage [55], which has been widely proposed as one mechanism of action for in vivo anti-tumor activity of GSIs. Other potential GSI targets whose inhibition could contribute to

AL101-induced sensitivity to paclitaxel include DCC, IGF1R, ErbB4, as well as numerous cytokine receptors including IL-6R and IL-1R.

GSIs target an additional class of enzymes called signal peptide peptidase (SPP) and SPP-like proteases (SPPL) that are inhibited by different GSIs to varying degrees [56]. SPP and SPPL also cleave single-pass membrane proteins, such as TNFa, MHC Class I preproteins, among a growing list of other substrates [57-60]. Thus, the mechanisms by which AL101 enhances the effects of paclitaxel in NSCLC could reach far beyond ɣ-secretase substrates. Identification of the targets responsible for GSI-induced sensitivity to chemotherapy would likely aide in the selection of the subgroup of NSCLC patients who will most benefit from the combined therapy.

Conclusion

Non-small cell lung cancer is a deadly disease for which there are an increasing number of, yet still too few, targeted therapies available. GSIs such as AL101 have been highly effective anti- cancer agents, as shown in numerous pre-clinical and clinical trials, even in some patients lacking overt driver mutations in the Notch pathway. We provided evidence that a selected subgroup of NSCLC tumors is exquisitely sensitive to AL101 plus paclitaxel. These findings support the need to continue the development of AL101 and other GSIs combined with cytotoxic chemotherapy for NSCLC patients. Validation studies are needed before we rule out Notch as the target of GSIs and biomarker for efficacy in NSCLC. We should also systematically test the myriad of other potential targets before continuing clinical trials in order to guide selection of the subset of patients who are most likely to benefit.

References

[1] Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin 2018;68:7-30.

[2] Malhotra J, Jabbour SK, Aisner J. Current state of immunotherapy for non-small cell lung cancer. Transl Lung Cancer Res 2017;6:196-211.

[3] Gulley JL, Rajan A, Spigel DR, et al. Avelumab for patients with previously treated metastatic or recurrent non-small-cell lung cancer (JAVELIN Solid Tumor): dose-expansion cohort of a multicentre, open-label, phase 1b trial. Lancet Oncol 2017;18:599-610.

[4] Pembrolizumab (Keytruda) https://www.fda.gov/Drugs/InformationOnDrugs/ApprovedDrugs/ucm558048.htm. 2017.

[5] Langer CJ, Gadgeel SM, Borghaei H, et al. Carboplatin and pemetrexed with or without pembrolizumab for advanced, non-squamous non-small-cell lung cancer: a randomised, phase 2 cohort of the open-label KEYNOTE-021 study. Lancet Oncol 2016;17:1497-508.

[6] Rotow J, Bivona TG. Understanding and targeting resistance mechanisms in NSCLC. Nature Reviews Cancer 2017;17:637.

[7] Capaccione KM, Pine SR. The Notch signaling pathway as a mediator of tumor survival. Carcinogenesis 2013;34:1420-30.

[8] Zhang Z, Nadeau P, Song W, et al. Presenilins are required for gamma-secretase cleavage of beta-APP and transmembrane cleavage of Notch-1. Nat Cell Biol 2000;2:463-5.

[9] O'Brien RJ, Wong PC. Amyloid Precursor Protein Processing and Alzheimers Disease. Annu Rev Neurosci 2011;34:185-204.

[10] Haapasalo A, Kovacs DM. The many substrates of presenilin/gamma-secretase. J Alzheimers Dis 2011;25:3-28.

[11] Haass C, Koo EH, Mellon A, Hung AY, Selkoe DJ. Targeting of cell-surface beta-amyloid precursor protein to lysosomes: alternative processing into amyloid-bearing fragments. Nature 1992;357:500-3.

[12] Shoji M, Golde TE, Ghiso J, et al. Production of the Alzheimer amyloid beta protein by normal proteolytic processing. Science 1992;258:126-9.

[13] Kumar R, Juillerat-Jeanneret L, Golshayan D. Notch Antagonists: Potential Modulators of Cancer and Inflammatory Diseases. J Med Chem 2016;59:7719-37.

[14] Doody RS, Raman R, Farlow M, et al. A Phase 3 Trial of Semagacestat for Treatment of Alzheimer's Disease. N Engl J Med 2013;369:341-50.

[15] Xia D, Watanabe H, Wu B, et al. Presenilin-1 Knockin Mice Reveal Loss-of-Function Mechanism for Familial Alzheimers Disease. Neuron 2015;85:967-81.

[16] Stump G, Durrer A, Klein AL, Lutolf S, Suter U, Taylor V. Notch1 and its ligands Delta-like and Jagged are expressed and active in distinct cell populations in the postnatal mouse brain. Mech Dev 2002;114:153-9.

[17] Dewachter I, Reverse D, Caluwaerts N, et al. Neuronal deficiency of presenilin 1 inhibits amyloid plaque formation and corrects hippocampal long-term potentiation but not a cognitive defect of amyloid precursor protein [V717I] transgenic mice. J Neurosci 2002;22:3445-53.

[18] Koo EH, Kopan R. Potential role of presenilin-regulated signaling pathways in sporadic neurodegeneration. Nat Med 2004;10 Suppl:S26-S33.

[19] Espinoza I, Miele L. Deadly crosstalk: Notch signaling at the intersection of EMT and cancer stem cells. Cancer Lett 2013;341:41-5.

[20] Andersson ER, Lendahl U. Therapeutic modulation of Notch signalling--are we there yet? Nat Rev Drug Discov 2014;13:357-78.

[21] Morgan KM, Shah JJ, Khiabanian H, Drake J, Pine SR. The gamma-secretase inhibitor BMS- 906024 sensitizes tumor cells to paclitaxel in non-small cell lung cancer. In Press ed. 2017. p. 1-6.

[22] Gao J, Aksoy BA, Dogrusoz U, et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci Signal 2013;6:l1.

[23] Comprehensive molecular profiling of lung adenocarcinoma. Nature 2014;511:543-50.

[24] Mehrad M, Roy S, Bittar HT, Dacic S. Next-Generation Sequencing Approach to NonSmall Cell Lung Carcinoma Yields More Actionable Alterations. Archives of Pathology & Laboratory Medicine 2017.

[25] Allen TD, Rodriguez EM, Jones KD, Bishop JM. Activated Notch1 induces lung adenomas in mice and cooperates with Myc in the generation of lung adenocarcinoma. Cancer Res 2011;71:6010- 8.

[26] Maraver A, Fernandez-Marcos PJ, Herranz D, et al. Therapeutic effect of gamma-secretase inhibition in KrasG12V-driven non-small cell lung carcinoma by derepression of DUSP1 and inhibition of ERK. Cancer Cell 2012;22:222-34.

[27] Licciulli S, Avila JL, Hanlon L, et al. Notch1 Is Required for Kras-Induced Lung Adenocarcinoma and Controls Tumor Cell Survival via p53. Cancer Res 2013.

[28] Yuan X, Wu H, Xu H, et al. Meta-analysis reveals the correlation of Notch signaling with non- small cell lung cancer progression and prognosis. Sci Rep 2015;5:10338.

[29] Capaccione KM, Hong X, Morgan KM, et al. Sox9 mediates Notch1-induced mesenchymal features in lung adenocarcinoma. Oncotarget 2014;5:3636-50.

[30] Hassan KA, Wang L, Korkaya H, et al. Notch pathway activity identifies cells with cancer stem cell-like properties and correlates with worse survival in lung adenocarcinoma. Clin Cancer Res 2013;19:1972-80.

[31] Simoes BM, O'Brien CS, Eyre R, et al. Anti-estrogen Resistance in Human Breast Tumors Is Driven by JAG1-NOTCH4-Dependent Cancer Stem Cell Activity. Cell Rep 2015;12:1968-77.

[32] Osipo C, Patel P, Rizzo P, et al. ErbB-2 inhibition activates Notch-1 and sensitizes breast cancer cells to a gamma-secretase inhibitor. Oncogene 2008;27:5019-32.

[33] Li JL, Sainson RC, Oon CE, et al. DLL4-Notch signaling mediates tumor resistance to anti-VEGF therapy in vivo. Cancer Res 2011;71:6073-83.

[34] Gavai AV, Quesnelle C, Norris D, et al. Discovery of Clinical Candidate BMS-906024: A Potent Pan-Notch Inhibitor for the Treatment of Leukemia and Solid Tumors. ACS Med Chem Lett 2015;6:523-7.

[35] Morgan KM, Fischer BS, Lee FY, et al. Gamma secretase inhibition by BMS-906024 enhances efficacy of paclitaxel in lung adenocarcinoma. Mol Cancer Ther 2017.

[36] Weijzen S, Rizzo P, Braid M, et al. Activation of Notch-1 signaling maintains the neoplastic phenotype in human Ras-transformed cells. Nat Med 2002;8:979-86.

[37] Ambrogio C, Gomez-Lopez G, Falcone M, et al. Combined inhibition of DDR1 and Notch signaling is a therapeutic strategy for KRAS-driven lung adenocarcinoma. Nat Med 2016;22:270-7.

[38] Skoulidis F, Byers LA, Diao L, et al. Co-occurring genomic alterations define major subsets of KRAS-mutant lung adenocarcinoma with distinct biology, immune profiles, and therapeutic vulnerabilities. Cancer Discov 2015;5:860-77.

[39] Arbour KC, Jordan E, Kim HR, et al. Effects of Co-occurring Genomic Alterations on Outcomes in Patients with KRAS-Mutant NonSmall Cell Lung Cancer. Clin Cancer Res 2018;24:334.

[40] Vasan N, Boyer JL, Herbst RS. A RAS Renaissance: Emerging Targeted Therapies for KRAS- Mutated NonÇôSmall Cell Lung Cancer. Clin Cancer Res 2014;20:3921.

[41] Westhoff B, Colaluca IN, D'Ario G, et al. Alterations of the Notch pathway in lung cancer. Proc Natl Acad Sci U S A 2009;106:22293-8.

[42] Kamstrup MR, Biskup E, Manf+¿ V, et al. Chemotherapeutic treatment is associated with Notch1 induction in cutaneous T-cell lymphoma. Leukemia & Lymphoma 2017;58:171-8.

[43] Debeb BG, Cohen EN, Boley K, et al. Pre-clinical studies of Notch signaling inhibitor RO4929097 in inflammatory breast cancer cells. Breast Cancer Res Treat 2012;134:495-510.

[44] Luistro L, He W, Smith M, et al. Preclinical profile of a potent gamma-secretase inhibitor targeting notch signaling with in vivo efficacy and pharmacodynamic properties. Cancer Res 2009;69:7672-80.

[45] Schott AF, Landis MD, Dontu G, et al. Preclinical and clinical studies of gamma secretase inhibitors with docetaxel on human breast tumors. Clin Cancer Res 2013;19:1512-24.

[46] Zhang CC, Pavlicek A, Zhang Q, et al. Biomarker and pharmacologic evaluation of the gamma- secretase inhibitor PF-03084014 in breast cancer models. Clin Cancer Res 2012;18:5008-19.

[47] Sanchez-Martin M, Ambesi-Impiombato A, Qin Y, et al. Synergistic antileukemic therapies in NOTCH1-induced T-ALL. Proc Natl Acad Sci U S A 2017;114:2006-11.

[48] Sanchez-Martin M, Ferrando A. The NOTCH1-MYC highway toward T-cell acute lymphoblastic leukemia. Blood 2017;129:1124-33.

[49] Stoeck A, Lejnine S, Truong A, et al. Discovery of biomarkers predictive of GSI response in triple- negative breast cancer and adenoid cystic carcinoma. Cancer Discov 2014;4:1154-67.

[50] Ohashi R, Takahashi F, Cui R, et al. Interaction between CD44 and hyaluronate induces chemoresistance in non-small cell lung cancer cell. Cancer Letters 2007;252:225-34.

[51] Okamoto I, Kawano Y, Murakami D, et al. Proteolytic release of CD44 intracellular domain and its role in the CD44 signaling pathway. J Cell Biol 2001;155:755.

[52] Miletti-Gonzalez KE, Murphy K, Kumaran MN, et al. Identification of function for CD44 intracytoplasmic domain (CD44-ICD): modulation of matrix metalloproteinase 9 (MMP-9) transcription via novel promoter response element. J Biol Chem 2012;287:18995-9007.

[53] McAuliffe SM, Morgan SL, Wyant GA, et al. Targeting Notch, a key pathway for ovarian cancer stem cells, sensitizes tumors to platinum therapy. Proc Natl Acad Sci USA 2012;109:E2939.

[54] Marambaud P, Shioi J, Serban G, et al. A presenilin-1/gamma-secretase cleavage releases the E- cadherin intracellular domain and regulates disassembly of adherens junctions. EMBO J 2002;21:1948-56.

[55] Kalen M, Heikura T, Karvinen H, et al. Gamma-Secretase Inhibitor Treatment Promotes VEGF-A- Driven Blood Vessel Growth and Vascular Leakage but Disrupts Neovascular Perfusion. PLoS One 2011;6:e18709.

[56] Mentrup T, Loock AC, Fluhrer R, Schroder B. Signal peptide peptidase and SPP-like proteases - Possible therapeutic targets? Biochim Biophys Acta 2017;1864:2169-82.

[57] Friedmann E, Hauben E, Maylandt K, et al. SPPL2a and SPPL2b promote intramembrane proteolysis of TNFalpha in activated dendritic cells to trigger IL-12 production. Nat Cell Biol 2006;8:843-8.

[58] Fluhrer R, Grammer G, Israel L, et al. A gamma-secretase-like intramembrane cleavage of TNFalpha by the GxGD aspartyl protease SPPL2b. Nat Cell Biol 2006;8:894-6.

[59] Lemberg MK, Bland FA, Weihofen A, Braud VM, Martoglio B. Intramembrane proteolysis of signal peptides: an essential step in the generation of HLA-E epitopes. J Immunol 2001;167:6441-6.

[60] Mentrup T, Fluhrer R, Schroder B. Latest emerging functions of SPP/SPPL intramembrane proteases. Eur J Cell Biol 2017;96:372-82.

Fig 1. Diagram depicting the ɣ-secretase complex and its cancer-related targets. Upper panel shows the ɣ-secretase complex, comprising PSEN, PEN2, APH1 and Nicastrin, which resides in the plasma membrane of lung tumor cells. The complex cleaves its substrates, Notch1-

4, CD44, and E-cadherin, among many other proteins. Lower panel shows the intracellular pro- tumorigenic signaling events caused by cleavage of the ɣ-secretase targets. Gamma-secretase

inhibitors (GSIs) inhibit the ɣ-secretase activity and prevent cleavage of the ɣ-secretase targets.

Rethinking gamma-secretase inhibitors for treatment of non-small cell lung cancer: Is Notch the target?

Sharon R. Pine

Clin Cancer Res Published OnlineFirst August 13, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-18-1635

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/early/2018/08/11/1078-0432.CCR-18-1635. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.