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Functional genomics for cancer and immune cell therapy

Introduction
The Peeper laboratory develops and uses function-based genomic approaches to better understand the mechanistic principles of cancer progression, and to identify novel therapeutic targets and predictive biomarkers for achieving more durable clinical responses for cancer patients. We have two main strategies. First, we want to increase our understanding of how cancer cells function and rewire their signaling networks. This will expose their weaknesses and allow or the identification of pharmacologically tractable vulnerabilities. Second, we are manipulating various cell types from the patient’s own immune system to enhance their specific cytotoxicity towards tumor cells. Also this approach should uncover new therapeutic targets, on immune cells. With these strategies, we aim to contribute to the development of combinatorial therapies, which simultaneously eliminate the patients’ tumor cells and harness their immune cells.

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Genetic profile of a human melanoma, demonstrating gene copy alterations

Function-based genomic screens
We use various experimental approaches to achieve these goals. A major tool in the laboratory is function-based, genome-wide screens: we have been developing and employing in vitro and in vivo genetic functional perturbation screens, for several cancer types, including melanoma and lung and breast cancer. Hits are identified in specific experimental settings allowing to identify essential gene functions in an efficient high-throughput manner. Candidates are analyzed by our own bioinformaticians. Often, their computational approaches provide additional insight into the signaling pathways affected by the screen hits. Eventually, identified genes are validated and characterized in-depth in a clinically relevant context, for example, patient-derived tumor xenografts (PDX) and humanized mouse models. The outcome of these strategies is the identification of druggable pathways as well as predictive biomarkers.

Clinical translation
The objectives outlined above imply that a central goal of our laboratory is to translate our findings to the benefit of the patient, taking advantage of our comprehensive cancer institute. To maximize these efforts, PI Daniel Peeper and Christian Blank (a clinician researcher) recently engaged in a partnership to complement our basic, translational and clinical fields of expertise. This warrants not only the clinical relevance of our research questions, but also facilitates translation of our laboratory findings (therapeutic targets, prognostic and predictive biomarkers) to the clinic, particularly by initiating trials that are run here at NKI.

PDX

An in vivo preclinical platform for melanoma
The therapeutic landscape of melanoma is improving rapidly. Targeted inhibitors show promising results, but drug resistance often limits durable clinical responses. There is a need for in vivo systems that allow for mechanistic drug resistance studies and (combinatorial) treatment optimization. Therefore, we established in collaboration with our clinical colleagues Haanen, Blank and Schumacher a large collection of PDX, derived from BRAFV600E, NRASQ61, or BRAFWT/NRASWT melanomas prior to treatment with BRAF inhibitor and after resistance had occurred. To demonstrate the utility of this platform, we took advantage of PDX as a limitless source and screened tumor lysates for new resistance mechanisms. We identified a BRAFV600E protein harboring a kinase domain duplication (BRAFV600E/DK) in ≈10% of the cases, both in PDX and in an independent patient cohort. While BRAFV600E/DK depletion restored sensitivity to BRAF inhibition, a pan-RAF dimerization inhibitor effectively eliminated BRAFV600E/DK-expressing cells. These results illustrate the value of this platform and warrant clinical validation of BRAF dimerization inhibitors and BRAFV600E/DK as a predictive biomarker for this group of melanoma patients.

Developing systems to integrate targeted and immunotherapy
Exciting advances have been made also for immunotherapy of melanoma, and indeed an increasing number of other cancer types. Several modes of activation are currently exploited to trigger patients’ own immune systems to allow for tumor eradication. Notwithstanding these clinical advances, it is clear that large groups of patients will not, or only temporarily, benefit from immunotherapy, mostly because of resistance. Therefore, in collaboration with the group of Ton Schumacher at NKI, we have built in vitro and in vivo systems to study tumor cell : T cell interactions. We use these systems to perform function-based screens to develop combinatorial targeted and immunotherapy regimens to achieve more durable clinical responses.

Similar matched epitope/TCR systems have now been set up for lung cancer, also to use large-scale genetic perturbations for the identification of predictive biomarkers and new therapeutic targets.

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Somatic mutations present in several melanoma metastases in a single patient, revealing that mutations were either shared by the pretreatment tumor and all metastases (blue), by some metastases (green), or only by single metastases (red)

Understanding and overcoming targeted drug resistance in melanoma
We previously found that the lack of the melanoma transcription factor MITF is associated with severe resistance to a range of targeted inhibitors. Both in intrinsic and acquired resistance, MITF levels inversely correlate with the expression of several activated receptor tyrosine kinases, most frequently AXL. The MITF-low/AXL-high/drug-resistance phenotype is common among mutant BRAF and NRAS melanoma cell lines. Drug cocktails containing AXL inhibitor enhanced melanoma cell elimination by BRAF or ERK inhibition. Our results demonstrate that a low MITF/AXL ratio predicts early resistance to multiple targeted drugs, and warrant clinical validation of AXL inhibitors to combat resistance of BRAF and NRAS mutant MITF-low melanomas. On the basis of these results, we engaged in a collaboration with the Dutch pharma company Genmab to explore clinical translation of these findings. For example, we are studying whether an AXL antibody-drug conjugate can serve as a new melanoma therapeutic.

Targeting cancer cell metabolism
We have previously discovered by metabolic profiling (icw. Eyal Gottlieb) and functional perturbations that the mitochondrial gatekeeper pyruvate dehydrogenase (PDH) is a crucial mediator of senescence induced by BRAFV600E. While the activation of PDH enhanced the use of pyruvate in the tricarboxylic acid cycle, causing increased respiration and redox stress, abrogation of oncogene-induced senescence (OIS) coincided with reversion of these processes. Enforced normalization of either PDK1 or PDP2 expression levels inhibited PDH and abrogated OIS, thereby licensing BRAFV600E-driven melanoma development. Depletion of PDK1 eradicated melanoma subpopulations resistant to targeted BRAF inhibition and caused regression of established melanomas. These results revealed a mechanistic relationship between BRAF OIS and identified a key metabolic signaling axis that may be exploited therapeutically. Recently, we have begun to functionally mine the metabolome for potential new therapeutic targets, both in tumor and immune cells.

Cancer drug addiction
Observations from cultured cells, animal models and patients raise the possibility that the dependency of tumors on the therapeutic drugs to which they have acquired resistance represents a vulnerability with potential applications in cancer treatment. However, for this drug addiction trait to become of clinical interest, we had to define the mechanism that underlies it. We performed an unbiased CRISPR–Cas9 knockout screen on melanoma cells that were both resistant and addicted to inhibition of BRAF, to functionally mine their genome for ‘addiction genes’. We discovered a signalling pathway comprising ERK2 and the JUNB/FRA1 transcription factor, disruption of which allowed addicted tumour cells to survive on treatment discontinuation, irrespective of the acquired drug resistance mechanism and which was conserved in ERGFRi-resistant lung cancer cells. As a PoC, we show that drug holiday synergized with chemotherapy. These results uncover a pathway that underpins drug addiction in cancer cells, which may help to guide the use of alternating therapeutic strategies for enhanced clinical responses in drug-resistant cancers.

Publications
For publications of the Peeper laboratory please click here.