Identification and modulation of drug targets for precision medicine in breast, lung and ovarian
cancer subtypes
Stutvoet, Thijs
DOI:
10.33612/diss.144705120
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2020
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Stutvoet, T. (2020). Identification and modulation of drug targets for precision medicine in breast, lung and
ovarian cancer subtypes. University of Groningen. https://doi.org/10.33612/diss.144705120
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Summary and future perspectives
SUMMARY
The treatment of cancer is composed of surgery, radiotherapy, and systemic therapy as a single modality or in combination. For advanced cancers, systemic therapies, such as chemotherapy, are the mainstay of treatment.1 Increased insight into cancer biology has enabled the development targeted agents and immunotherapy.2,3 While chemotherapy targets all dividing cells, targeted therapies inhibit tumor cell-specific alterations.2,3 Targeted agents have improved the treatment of patients with tumors harboring targetable alterations, such as epidermal growth factor receptor (EGFR) mutations and epidermal growth factor receptor 2 (HER2) overexpression.4,5 Immunotherapy induces an anti-tumor immune response, improving overall survival across several tumor types.6 However, many cancer patients do not yet benefit of these new treatments. This may be caused by several factors, including a lack of targetable alterations, a lack of approved inhibitors for those tumor-specific alterations, or resistance to therapy.7 Uncovering novel targets, selecting the right patient for the right treatment, and combining multiple agents may improve the impact of targeted- or immunotherapy.
The research presented in this thesis aimed to provide a biological rationale for potential combination therapies and patient selection tools to enhance efficacy of immunotherapy and targeted therapies in patients with subtypes of breast cancer, non-small cell lung cancer (NSCLC) and ovarian cancer.
Breast cancer consists of four subtypes with different biological characteristics and treatment options. In chapter 2, we describe a literature study that was published in
2015, regarding the opportunities and advances of immunotherapy in breast cancer at that time. The HER2+ and triple negative breast cancer (TNBC) subtypes harbor the highest mutational load, presumably making them more immunogenic. Subsequently, in these subtypes tumor infiltrating lymphocytes (TILs) are related to overall survival and predict response to anthracyclines. Preclinical studies indicate important immune-based mechanisms of chemotherapeutics, in particular of anthracyclines, and HER2-targeting antibodies that partially depend on natural killer cells for their effects. In 2015 immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1), programmed cell death protein 1 ligand (PD-L1), and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) appeared to have some anti-tumor efficacy in patients with breast cancer. Tumor vaccines induce immunity against a range of tumor-specific targets, such as HER2, mammaglobin-A, and MUC-1. However, their clinical efficacy has not yet been demonstrated. Novel strategies, including bispecific T cell engagers, dendritic cell engagers, and chimeric antigen receptor (CAR) T cells are being tested in breast cancer. Concluding, although
only small clinical trials had looked into the efficacy of immunotherapy in breast cancer, in 2015 there were signs of potential use in patients with HER2+ breast cancer and TNBC. Immune checkpoint inhibitors targeting PD-1 and PD-L1 have improved the survival of patients with NSCLC. Still, many patients do not respond to these inhibitors. PD-L1 expression is dynamic and is related to the efficacy of immune checkpoint inhibitors. In chapter 3, we studied the regulation of PD-L1 expression in NSCLC without targetable genetic
alterations. Analysis of RNA sequencing data from these NSCLCs revealed that inferred interferon gamma (IFNγ), EGFR, and mitogen-activated protein kinase (MAPK) signaling correlated with PD-L1 gene expression in tumors of patients with adenocarcinomas. In a representative lung adenocarcinoma cell line panel, stimulation with epidermal growth factor (EGF) or IFNγ increased PD-L1 mRNA, protein, and membrane levels, which were further enhanced by combining EGF and IFNγ. Similarly, tumor cell PD-L1 membrane levels increased after coculture with activated peripheral blood mononuclear cells from healthy volunteers. Inhibition of the MAPK pathway, using EGFR inhibitors cetuximab and erlotinib or the mitogen-activated protein kinase kinase 1 and 2 (MEK1/2) inhibitor selumetinib, prevented the induction of PD-L1 mRNA, protein and membrane expression by EGF and IFNγ, but did not affect IFNγ-induced MHC-I upregulation. Interestingly, while IFNγ induced PD-L1 expression through transcription, MAPK signaling additionally increased PD-L1 mRNA stability. In conclusion, MAPK pathway activity plays a key role in EGF- and IFNγ-induced PD-L1 expression in lung adenocarcinoma without targetable genetic alterations and may present a target to improve the efficacy of immunotherapy.
18F-BMS-986192, an adnectin-based human PD-L1 targeting tracer, was developed for same-day PD-L1 PET imaging. In chapter 4, we evaluated the ability of 18F-BMS-986192 PET to detect different PD-L1 expression levels and therapy-induced changes in PD-L1 expression. Binding of 18F-BMS-986192 correlated with PD-L1 membrane expression in tumor cell lines. In addition, 18F-BMS-986192 tumor tracer uptake in mice bearing human xenografts was associated with PD-L1 expression measured immunohistochemically. IFNγ treatment increased PD-L1 expression in tumor cell lines and caused up to 12-fold increase in tracer binding. In vivo, IFNγ did neither affect PD-L1 tumor expression measured immunohistochemically nor 18F-BMS-986192 tumor uptake. In vitro, selumetinib reduced tumor cell cellular and membrane levels of PD-L1 with 50%. In mice, selumetinib lowered cellular, but not membrane PD-L1 levels in tumors. Consequently no treatment-induced changes in 18F-BMS-986192 tumor uptake were observed. Concluding, 18F-BMS-986192 PET imaging allows detection of membrane-expressed PD-L1, as soon as 60 minutes after tracer injection. The first studies with this tracer in patients have already been performed.
The addition of poly-ADP ribose polymerase (PARP) and angiogenesis targeting agents to treatment with chemotherapy has marginally improved overall survival of patients with advanced high grade serous ovarian cancer (HGSOC). Gene expression profiles of HGSOCs increased understanding of tumor biology, but have not resulted in novel treatment strategies. These expression profiles represent the average expression of all cancerous and non-cancerous cells present in the biopsy. Therefore, the transcriptional footprint of relevant biological processes may be overshadowed by other transcriptional footprints, making them difficult to detect. In chapter 5, we applied consensus -
independent component analysis (c-ICA) on expression profiles of 1,089 epithelial ovarian cancer samples to identify subtle transcriptomic processes related to clinical endpoints. We identified 374 distinct transcriptional footprints and related those to biological processes using gene set enrichment analysis (available at http://83.162.250.102/ TranscriptionalLandscapeOvarianCancer/). Six transcriptional footprints classified patients with platinum-treated serous ovarian cancer into distinct survival cohorts. These transcriptional footprints were associated with 1) neuronal development, 2) replication & apoptosis due to alterations in chromosome 13q12-q14, 3) proliferation & immune response due to alterations in chromosome 11q13-q15, 4) replication stress due to alterations on chromosome 9p13-p21, 5) neurotransmitter signaling, and 6) extracellular matrix interaction. The cohort with the worst survival (15% of the patients) was defined by high activity of the transcriptional footprint with similarities to neuronal development. This study shows the strength of independent component analysis in dissecting gene expression datasets and unveiled biological processes relevant for the clinical outcome of HGSOC.
Advanced ovarian clear cell cancer (OCCC) is characterized by platinum-resistance and worse patient survival compared to other epithelial ovarian cancer subtypes, including HGSOC. Interestingly, around 50% of OCCC tumors harbor AT-rich Interaction Domain 1A (ARID1A) mutations. In chapter 6, we studied the ataxia telangiectasia and rad 3
related (ATR) – checkpoint kinase 1 (CHK1) DNA damage response pathway as a target in OCCC. Gene expression profiles of OCCC tumors (n = 67) were enriched for DNA repair pathway genes compared to healthy ovarian surface epithelium (n = 22). Next, we studied sensitivity to inhibitors of ATR (VE821) and CHK1 (Chir-124) of 8 OCCC cell lines, including
ARID1A wild-type, mutant, and knockout cell lines. The ARID1A status was not related to
sensitivity for cisplatin or VE821 and Chir-124 in short- and long-term cell survival assays. VE821 and Chir-124 sensitized both ARID1A wild-type and mutant cells to cisplatin. These combination treatments increased the amount of γH2AX+ cells compared to single agent treatment, indicating an increase in double strand DNA breaks. Concluding, ATR-CHK1 pathway inhibition sensitizes OCCC cells to cisplatin independent of ARID1A status.
DISCUSSION AND FUTURE PERSPECTIVES
The introduction of immune checkpoint inhibitors into clinical practice has extended the range of therapeutic options for advanced cancers.6 They have increased survival of patients with aggressive tumor types, including melanoma, NSCLC and bladder cancer.6 In patients with metastatic melanoma immunotherapy induces durable complete responses and a subgroup may even be cured.8 Responses to inhibitors of oncogenic drivers, such as EGFR, HER2 and BRAF are often short-term, due to secondary resistance mechanisms like mutations in the tyrosine kinase domain or compensatory activation of alternative signaling pathways.3,9 The mechanism of action and resistance mechanisms of some of these inhibitors are relatively well understood, and inhibitors that specifically target resistant cells have been developed.9 The mechanism of action of immune checkpoint inhibitors have not been completely elucidated.6 Expression of target proteins is not the only determinant of treatment response. In NSCLC immune checkpoint inhibitors are now standard of care. But even in patients with PD-L1 positive NSCLC, 55% does not respond to PD-1 targeted antibodies.10 Enhanced insight in the biological mechanisms of immune checkpoint inhibitors may pave the way for novel therapeutic combination strategies and improve patient selection.
Since the publication of our review on immunotherapy in breast cancer in 2015 (chapter 2), immunotherapy has emerged as a viable treatment option in TNBC. Translational
research revealed that TNBC has the highest levels of neo-antigens of all breast cancer subtypes. In addition, a direct link between DNA damage and pro-inflammatory signaling through protein cyclic GMP-AMP synthase – stimulator of interferon genes (cGAS-STING) provides a rationale for the use of immunotherapy in TNBC.11,12 Subsequently, clinical studies were performed with immune checkpoint inhibition in TNBC. Single-agent immune checkpoint inhibition has modest activity, with PD-1 inhibitor pembrolizumab achieving an objective response rate of only 5.3% in 170 previously treated patients with TNBC.13 In patients with TNBC with more than 10% PD-L1 positive cells in the tumor microenvironment, response rates to first-line pembrolizumab were better (21.4%), but progression free survival (PFS) remained disappointing (2.1 months). Because chemotherapeutic drugs enhance cGAS-STING signaling and immunogenicity of cancer cells, trials combining chemo- and immunotherapy have been performed.14 In 902 previously untreated patients with PD-L1 positive advanced TNBC adding PD-L1 antibody atezolizumab to paclitaxel improved median survival from 15.5 to 25 months.15 This study resulted in approval of atezolizumab for clinical use.16 Combinations with platinum-based chemotherapy have been studied in a phase II trial in patients with advanced TNBC. Cisplatin activated the PD-1/PD-L1 pathway, increased expression of genes related to T
cell cytotoxicity in the tumor microenvironment, and enhanced response rates to PD-1 inhibitor nivolumab.17 Moreover, the PD-1 inhibitor pembrolizumab has already been approved by the FDA in combination with chemotherapy in the neoadjuvant setting. Additional studies looking into the interplay between chemotherapy and immune cell activation are currently being performed.18–20 Phase II and III trials looking into the efficacy of immune checkpoint inhibitors as monotherapy21,22 or in combination with chemotherapy23,24 are ongoing in the neo-adjuvant and adjuvant setting. Moreover, studies are evaluating the combination of immune checkpoint inhibitors with PARP, histone deacetylase (HDAC) and cyclin dependent kinase (CDK) inhibitors in TNBC and ER+ breast cancer.25–28
HGSOC has many overlapping characteristics with TNBC, including the high frequency of P53 mutations, deficiencies in homologous recombination, and copy number alterations.29 This might suggest that immunotherapy can also be effective in HGSOC. However, single agent PD-1 / PD-L1 targeting immunotherapies achieved response rates of only 5.9 – 15% in pretreated advanced HGSOC.30–33 Patient selection may be needed to improve response rates.34 For example, selection for atezolizumab in patients with metastatic TNBC is currently based on immunohistochemical analysis of PD-L1 expression in the tumor.15 Meta-analysis of HGSOC tumors showed PD-L1 expression in the tumor microenvironment of 58% of these tumors, suggesting PD-1 / PD-L1 blockade may be an active drug in a subset of patients with HGSOC.35 Amplifications of PD-L1 can also predict response to therapy, as is observed in Hodgkin’s lymphoma.36 In patients with melanoma treated with pembrolizumab, the pretreatment number of intratumoral CD8+ T cells is higher in responders compared to non-responders. These CD8+ T cell numbers further increase during treatment.37 Ovarian cancer tumors are generally seen as an ‘immune desert’. In a cohort of 5,500 patients with ovarian cancer, however, HGSOC tumors harbored relatively high numbers of TILs compared to other ovarian cancer subtypes.38,39 Moreover, the number of CD8+ T cell infiltration in these tumors was strongly related to overall survival in chemotherapy-treated patients with HGSOC. Combined analyses of immune checkpoint proteins and TILs may enhance the predictive value for immune checkpoint inhibitor efficacy.40 Across cancer types, tumor mutational burden is a factor that can support the prediction of response to immune checkpoint inhibition.41 This is not the case in HGSOC. Interestingly, breast cancer gene (BRCA) 1 and 2 mutant HGSOC tumors have higher levels of TILs, which express more PD-1 and PD-L1, compared to other HGSOC tumors.12,42,43 BRCA status may thus be used to find patients with more immunogenic tumors. Currently, immunohistochemical analyses of a single biopsy are mostly used to study TIL infiltration and PD-L1 expression. However, this may cause sampling errors due to the dynamic and heterogeneous nature of both
TIL levels and PD-L1 expression.44–46 Whole body PET imaging of immunological targets can be used to infer whole-body immune checkpoint protein or immune cell presence.47 A recent study in 22 patients with bladder cancer, NSCLC, and TNBC showed that PD-L1 PET tracer uptake had better predictive value than immunohistochemical measurement of tumor PD-L1 expression.48 Further development and validation of these tracers may allow their implementation for PET imaging into the clinic.
Standard treatment of ovarian cancer includes platinum-based chemotherapy, which can increase tumor immunogenicity by inducing immunogenic cell death.49 These treatments may also, partially, depend on pretreatment immune cell activation for their efficacy. For example, the addition of carboplatin to neo-adjuvant anthracyclines and taxanes improved response only in patients with high levels of stromal TILs.50 In advanced ovarian cancer neo-adjuvant platinum-based chemotherapy also increased the number of TILs with 20-30%. This was accompanied by increased tumoral PD-L1 expression.51 In syngeneic mouse models of head and neck cancer, cisplatin sensitized tumors to PD-L1 antibodies by increasing antigen presentation and T cell killing.52 Combining immunotherapy with chemotherapy has been attempted in HGSOC, but efficacy was disappointing. Even in patients with PD-L1 positive tumors, the addition of PD-L1 blocking antibody atezolizumab to liposomal doxorubicin did not improve PFS or overall survival.39 In addition, in patients with advanced ovarian cancer, the addition of avelumab to first-line platinum-based chemotherapy was stopped after an interim analysis indicated no improved PFS (18.1 vs 16.8 months, NCT02718417). These disappointing results may be explained by the fact that HGSOC tumors harbor a relatively low mutational burden compared to cancer types where immune checkpoint inhibition is more effective.53 Moreover, HGSOC is characterized by copy number alterations, which are associated with reduced response to immune checkpoint inhibition.54,55 One of the factors related to tumor-induced immune suppression is the production of vascular endothelial growth factor (VEGF). VEGF directly suppresses effector cells of the adaptive and innate immune system, causes a hypoxic and immune-suppressive tumor microenvironment and has a systemic immune suppressive function.56 In ovarian cancer, combining VEGF inhibition with PD-1 blockade improves response rates more in patients with platinum-sensitive disease compared to patients with platinum-resistant disease (40% objective response rate vs 16.7%).57 A second study in nine patients with HGSOC, in which PD-1 inhibition was combined with a VEGF kinase receptor inhibitor, induced responses in 55% of patients. However, seven of these patients experienced grade 2-4 adverse events, and one patient died due to a pulmonary embolism.58 In
chapter 3, we suggest inhibition of the MAPK pathway as a strategy to improve the
immunogenicity of NSCLC cells. Moreover, in chapter 4, these findings are translated
to an in vivo setting and BRAF mutant cancer. For HGSOC, it is unlikely that targeting the MAPK pathway will have large antitumor effects, as it does not play a major role in this cancer type. However, genomic instability is supposed to play an important role in tumorigenesis.59 Moreover, in HGSOC tumors DNA damage repair deficiencies relate to increased immune cell activation.43,60 This observation has led to the hypothesis that immune cell reactivity in HGSOC may be driven by DNA damage response signaling.61 In preclinical models, PARP inhibitors and other DNA damaging agents activate the cGAS-STING pathway, increase the tumor immune response, and improve the efficacy of immune checkpoint inhibitors.62–65 In analogy to these findings, ATR pathway inhibition may not only enhance cisplatin cytotoxicity in OCCC (chapter 6), but also enhance
immunogenicity of OCCC.66 Combining PARP inhibitors with PD-1 antibodies in patients with homologous recombination proficient platinum resistant advanced HGSOC resulted in response rates of 17-18%.58,67 Preselecting HGSOC with mutant BRCA1 improved response rates to PARP and immune checkpoint inhibition to 63%.68 Multiple studies combining DNA damaging agents with immunotherapy are currently ongoing in patients with HGSOC.39 The results of these studies may provide insight in which patients should when be considered for such treatments, and what the best combinations and sequence of treatments are.
Immunotherapy remains an undelivered promise in HGSOC. Despite the induction of long-lasting responses in several patients, the overwhelming majority of patients derives no clinical benefit from immune checkpoint inhibitors in the first-line and recurrent setting. DNA damaging agents, such as PARP inhibitors or platinum-based chemotherapy may prime the anti-tumor immune response and improve the efficacy of immunotherapy. However, optimal combination strategies, timing of these interventions, and patient selection criteria remain to be determined.
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