Notwithstanding clinical advances, it is clear that large groups of patients will not durably benefit from immunotherapy, mostly because of resistance. Therefore, in collaboration with the group of Ton Schumacher, we have built in vitro and in vivo systems to study interactions between tumor cells and T cells. We have used these systems to perform function-based screens to develop combinatorial targeted and immunotherapy regimens to achieve more durable clinical responses.
For example, we have used this approach to break intrinsic resistance of melanoma to T cell killing. Whereas the interferon ((IFN) pathway harbors both ICB resistance factors and therapeutic opportunities, this has not been systematically investigated for (IFN)γ -independent signaling routes. A genome-wide CRISPR/Cas9 screen to sensitize IFNγ receptor-deﬁcient tumor cells to CD8 T cell elimination uncovered several hits mapping to the tumor necrosis factor (TNF) pathway. Clinically, we have shown that TNF antitumor activity is only limited in tumors at baseline and in ICB non-responders, correlating with its low abundance. Taking advantage of the genetic screen, we have also demonstrated that ablation of the top hit, TRAF2, lowers the TNF cytotoxicity threshold in tumors by redirecting TNF signaling to favor RIPK1-dependent apoptosis. TRAF2 loss greatly enhanced the therapeutic potential of pharmacologic inhibition of its interaction partner cIAP, another screen hit, thereby cooperating with ICB. Our results suggest that selective reduction of the TNF cytotoxicity threshold increases the susceptibility of tumors to immunotherapy.
Continuing on this theme, we considered that little is known about mechanisms of intrinsic immune resistance. To mimic recurrent T cell attack, we chronically exposed a panel of (patient-derived) melanoma cell lines to cytotoxic T cells. This led to strong enrichment of a pre-existing cell population that exhibited immune resistance in vitro and in mice. These fractions showed high expression of NGFR, were maintained stably, and were found to be present in patients’ melanomas prior to treatment. Remarkably, these cells exhibited multidrug-resistance to other therapies including BRAF + MEK inhibition, suggesting that they exist in a stable and distinct cellular state. We are currently clinically corroborating these findings, and exploring how to translate this findings therapeutically.
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 allowing for mechanistic drug resistance studies and (combinatorial) treatment optimization. Therefore, we established in collaboration with our clinical colleagues prof. Haanen, Blank and Schumacher a large collection of PDX, derived from BRAFV600E, NRASQ61, or BRAFWT/NRASWT melanoma metastases prior to treatment with BRAF inhibitor and after resistance had occurred. We have demonstrated the utility of this platform both for discovery of resistance mechanisms and preclinical validation of new treatments. We have recently derived a few dozen low-passage cell lines that we are currently characterizing, also in the context of immuno-oncology.
We found that the lack of the melanoma transcription factor MITF expression is associated with severe resistance to a range of precision medicines targeting the BRAF pathway. Both in intrinsic and acquired therapy 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. In a follow-up study, we recently showed that intratumor heterogeneity is a key factor contributing to therapeutic failure and, hence, cancer lethality. Heterogeneous tumors show partial therapy responses, allowing for the emergence of drug-resistant clones that often express high levels of the receptor tyrosine kinase AXL. In melanoma, AXL-high cells are resistant to MAPK pathway inhibitors, whereas AXL-low cells are sensitive to these inhibitors, rationalizing a differential therapeutic approach. We collaborated with Genmab, which developed an antibody-drug conjugate, AXL-107-MMAE, comprising a human AXL antibody linked to a microtubule-disrupting agent. AXL- 107-MMAE displayed potent in vivo anti-tumor activity in PDX, including melanoma, lung, pancreas and cervical cancer. By eliminating distinct populations in heterogeneous melanoma cell pools, AXL-107-MMAE and MAPK pathway inhibitors cooperatively inhibited tumor growth. Furthermore, by inducing AXL transcription, BRAF/MEK inhibitors potentiated the efficacy of AXL-107-MMAE. These findings provide proof of concept for the premise that rationalized combinatorial targeting of distinct populations in heterogeneous tumors may improve therapeutic effect, and merit clinical validation of AXL-107-MMAE in both treatment-naive and drug-resistant cancers in mono- or combination therapy.
We discovered that cancers can get addicted to the very drugs that serve to eliminate them. With a CRISPR–Cas9 knockout screen, we uncovered a signalling pathway comprising ERK2 and JUNB/FRA1 transcription factors, disruption of which allowed addicted tumour cells to survive on treatment discontinuation. In patients with melanoma that had progressed during treatment with a BRAF inhibitor, treatment cessation was followed by increased expression of the receptor tyrosine kinase AXL, which is associated with a phenotype switch. Drug discontinuation synergized with the melanoma chemotherapeutic agent dacarbazine by further suppressing MITF and its prosurvival target BCL-2, and by inducing DNA damage in cancer cells. 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.
The objectives outlined above illustrate 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, Daniel Peeper and Christian Blank (a clinician researcher/medical oncologist) have engaged in a partnership to complement their respective basic and clinical 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.