Repurposing anticancer drugs for targeting necroptosis

ABSTRACT

Necroptosis represents a form of programmed cell death that can be engaged by various upstream signals, for example by ligation of death receptors, by viral sensors or by pattern recognition receptors. It depends on several key signaling proteins, including the kinases Receptor-Interacting Protein (RIP)1 and RIP3 and the pseudokinase mixed-lineage kinase domain-like protein (MLKL). Necroptosis has been implicated in a number of physiological and pathophysiological conditions and is disturbed in many human diseases. Thus, targeted interference with necroptosis signaling may offer new opportunities for the treatment of human diseases. Besides structure-based drug design, in recent years drug repositioning has emerged as a promising alternative to develop drug-like compounds. There is accumulating evidence showing that multi-targeting kinase inhibitors, for example Dabrafenib, Vemurafenib, Sorafenib, Pazopanib and Ponatinib, used for the treatment of cancer also display anti-necroptotic activity. This review summarizes recent evidence indicating that some anticancer kinase inhibitors also negatively affect necroptosis signaling. This implies that some cancer therapeutics may be repurposed for other pathologies, e.g. ischemic or inflammatory diseases.

Introduction

Tissue homeostasis in multicellular organisms is maintained by a balance between cell death on one side and cell growth on the other side [1]. Programmed cell death is a cell-intrinsic process that plays a central role in various physiological and pathophysiological conditions [1]. While apoptosis was once considered as one of the most relevant forms of programmed cell death, the identification of additional modes of mammalian programmed cell death has changed this view in recent years [2]. Necrosis has for a long time been considered exclusively as an accidental mode of cell death without any underlying coordinated program of signaling events. More recently, breakthrough discoveries have shown that regulated forms of necrosis also exist [3]. For example, necroptosis is a mode of programmed cell death that is coordinated by defined signaling events [4]. This characteristic feature of concerted signaling events opens up the possibility for targeted intervention, e.g. for therapeutic purposes. Since there is mounting evidence showing that necroptosis is often dysregulated in many human diseases such as ischemic organ damage and inflammatory disorders [5], there is a growing interest to selectively target necroptosis signaling for developing new treatment approaches. To this end, a number of drug design and development efforts have led to the identification of small-molecule tool compounds that target key proteins of the necroptosis signaling pathway [6].

In addition to these drug design efforts, drug repositioning of compounds that have already been approved by the Food and Drug Administration (FDA) offers an attractive alternative approach. Since the development and the approval of new drugs represent a costly and arduous process that also has a high failure rate, there is growing interest in drug repurposing. Drug repurposing implies the use of existing compounds that have already been tested in humans and have shown a level of tolerability and safety acceptable for further investigation for a medical condition other than originally designed [7]. Such drugs often have identified molecular targets and medical indications for which they have already been approved or have been sidelined for various reasons somewhere along the pipeline [8]. One of the key advantages of drug repurposing resides in the fact that the development track may avoid unexpected failures not predicted by preclinical data, for example due to toxicities. Drug repurposing may imply the identification of novel molecular targets with separate or new indications for the same target.

As far as drug repurposing in the field of necroptosis is concerned, several anticancer drugs have been identified in recent years to impinge, besides their known targets, also on components of the necroptotic machinery. This review summarizes some key issues of the opportunities and challenges of recent discoveries showing that some anticancer therapeutics may have an impact on necroptosis signaling.

Necroptosis signaling

Key signaling molecules of necroptosis comprise the serine/threonine kinases RIP1 and RIP3 and the pseudokinase MLKL. One of the best characterized necroptosis signaling cascades so far is engaged upon binding of Tumor Necrosis Factor (TNF)α, a member of the death receptor ligand superfamily, to its cognate receptor TNFR1 on the cell surface [3]. This leads to receptor internalization, assembly of RIP1 together with RIP3 into the necrosome complex and activation of RIP3 [3]. In turn, activated RIP3 stimulates activation of MLKL, which oligomerizes and translocates to the plasma membrane where it forms pores and disrupts the plasma membrane integrity [3]. Posttranslational modifications such as phosphorylation play an important role in regulating signal transduction. In the course of necroptosis, consecutive phosphorylation events involving RIP1, RIP3 and MLKL are critical for signal transmission [3]. Accordingly, activated RIP1 phosphorylates and activates RIP3, which in turn activates MLKL by phosphorylation [3].

Discovery of anticancer drugs targeting the necroptosis pathway

It is well-known that there is a substantial similarity in the kinase domains of many human kinases. This also applies to the kinase domains of RIP1 and RIP3. For example, the kinase domains of both RIP1 and RIP3 display a high degree of sequence similarity with the kinase domain of B-RAF [9]. Structural biology studies comparing inhibitor-bound RIP1 to inhibitor-bound B-RAF revealed several binding sites that partially overlap for pharmacological inhibitors of RIP1 (i.e. Necrostatin-1) and B-RAF (i.e. Vemurafenib) [9]. This similarity suggests that inhibitors that are directed against the oncogenic kinase B-RAF may also inhibit the kinase activities of RIP1 and/or RIP3. These insights gained from structural biology have provided the necessary background to test pharmacological inhibitors of B-RAF in models of necroptosis. Indeed, B-RAF inhibitors including Dabrafenib and Vemurafenib have been reported to block RIPK3 [10]. These anti-necroptotic activities in cellular models of necroptosis have occurred independently of their inhibitory effects on B-RAF [10]. Among a range of six different B-RAF inhibitors tested, Dabrafenib turned out to offer the most potent inhibition on RIP3, which was achieved by its ATP-competitive binding to the enzyme [10]. Importantly, in acetaminophen-overdosed mouse models, Dabrafenib was found to mitigate toxin-triggered liver damage [10]. In vitro, Dabrafenib prevented acetaminophen-induced necrosis in normal human hepatocytes, which is considered to be mediated by RIP3 [10]. In vivo using an acetaminophen-overdosed mouse model, Dabrafenib was found to ease the acetaminophen-caused liver damage and to prevent acetaminophen-induced necrosis in normal human hepatocytes, a type of cell death that is considered to be mediated by RIP3 [10].

In addition to B-RAF inhibitors, Sorafenib has very recently been identified as another anticancer drug that can inhibit necroptosis [11,12]. Sorafenib is a multi-targeting tyrosine kinase inhibitor that has been shown to inhibit B-RAF in addition to other kinases including VEGF, PDGF, FLT3 receptor and c-KIT [13]. Sorafenib is used as an anticancer agent, for example in the treatment of acute myeloid leukemia (AML), hepatocellular carcinoma (HCC) and advanced renal cell carcinoma [13,14]. Martens et al. identified Sorafenib as an inhibitor of necroptosis using a high-content screening of FDA-approved drug libraries and small compounds [11]. Sorafenib has been demonstrated to block kinase activity of both RIPK1 and RIPK3 [11]. In pull-down experiments, biotinylated Sorafenib has been found to directly interact with RIPK1, RIPK3 and MLKL [11]. Consequently, Sorafenib rescued murine as well as human cell lines from TNFα-stimulated necroptosis [11]. Also, Sorafenib has been described to protect apoptosis-resistant AML cells from Second mitochondria-derived activator of caspases (Smac) mimetic-induced necroptosis, including primary, patient-derived AML blasts [12]. This Sorafenib-conferred protection occurred at subtoxic, sub- to low micromolar concentrations of Sorafenib corresponding to plasma levels that were reported in cancer patients [11,12], emphasizing the clinical relevance of these findings. At higher concentrations or upon prolonged treatment, Sorafenib caused cell death, pointing to a concentration- and time-dependent dual activity spectrum [11,12]. Importantly, Sorafenib has been shown to provide protection in two in vivo models of necroptosis, that is in renal ischemia-reperfusion injury and in TNF-induced systemic inflammatory response syndrome [11]. Together, these findings demonstrate that Sorafenib exerts potent in vitro and in vivo anti-necroptotic activities.

Furthermore, a cellular screen with FDA-approved drugs identified Pazopanib and Ponatinib as necroptosis inhibitors that suppressed necroptosis in human cells at submicromolar EC50 concentrations [15]. In an independent screen, Ponatinib has also been identified as an inhibitor of necroptosis that was stimulated by structural homology of RIP1 and ABL [16], as the Glu-in/DLG-out conformation of RIPK1 is similar to that of ABL [17].

Pazopanib is a multi-targeting receptor tyrosine inhibitor of VEGFR1/2/3, PDGFR, FGFR, c-KIT and c-Fms and is approved for the treatment of patients with advanced renal cell carcinoma and soft-tissue sarcoma [18,19]. In studies aiming at elucidating the mode of the anti-necroptotic action of Pazopanib, RIPK1 has been identified as the main functional target of Pazopanib, whereas the established targets of Pazopanib turned out to be dispensable [15]. The potential clinical relevance of the identification of Pazopanib as an inhibitor of necroptosis is underlined by data showing its protective effect against necroptosis at submicromolar concentrations (1-5 micromolar) [15], which is well below plasma concentrations that were reported to be achieved in cancer patients in the clinic (between 20 and 40 μM) [20,21]. In contrast to the ability of Pazopanib to block necroptosis, the inhibitor failed to interfere with apoptosis [15].

Ponatinib is known as a pan-BCR-ABL tyrosine kinase inhibitor and approved for the treatment of chronic myeloid leukemia (CML) [22,23]. Ponatinib was found to block necroptosis at submicromolar concentrations, being even more potent in blocking necroptosis than Necrostatin-1s that is widely used as a RIPK1 inhibitor [15]. Target profiling by drug affinity purifications revealed that Ponatinib but no other BCR-ABL inhibitors directly targets both RIPK1 and RIPK3 [15]. This dual targeting of RIPK1 and RIPK3 by Ponatinib has been demonstrated in an independent study [16]. Concomitant inhibition of both RIPK1 and RIPK3 may have potential implications, for example for targeting pathophysiological conditions that are driven by both RIPK1 and RIPK3. The Ponatinib scaffold has also been utilized to develop new types of RIP1 inhibitors with improved profiles [16]. These Ponatinib derivatives not only potently protected cells from necroptosis in vitro, but also from TNFα-induced injury in vivo [16].

In addition, Ponatinib has been found to bind Transforming growth factor beta-activated kinase (TAK)1 with high affinity [15]. TAK1 has recently been reported to drive RIPK3-dependent necrosis upon TNFα challenge besides its well-established function in NF-κB activation [24]. From a translational perspective, it is relevant to note that the effective drug concentrations of Ponatinib that were required for blocking necroptosis are within the concentration range (120-140 nM) reported in the plasma of patients upon administration of the recommended dose [23]. In contrast to inhibiting necroptosis, Ponatinib did not interfere with the induction of apoptosis [15].

Conclusions and Discussion

Together, these studies imply that several FDA-approved anticancer drugs, including Dabrafenib, Vemurafenib, Sorafenib, Pazopanib and Ponatinib, may be repurposed for the treatment of other diseases than cancer that are driven in a RIP1- and/or RIP3-dependent manner. This applies to diseases such as ischemia-reperfusion injury or systemic inflammatory response syndrome that involve inflammation and tissue injury.

Targeting RIP3 may offer the advantage of rescuing cells from a wider spectrum of necroptotic stimuli compared to RIPK1 inhibition, as necroptosis may also occur in a RIP1- or RIP1 kinase activity-independent manner. However, drug design of RIP3 inhibitors needs to take into account that not only selective small-molecule RIPK3 inhibitors but also a kinase-dead variant of RIPK3, i.e. RIPK3 D161N, has been described to engage caspase-8-dependent apoptosis [25,26]. Despite these concerns as to whether or not targeting RIPK3 kinase has a therapeutic potential, it may in principle be feasible to exploit RIP3 as a therapeutic target for blocking cell death based on data showing that kinase-dead RIP3K51A/K51A mice develop normally, whereas the RIPK3 D161N mutation is embryonically lethal [25,26].

Repositioning already existing drugs for new indications is considered as a promising approach to deliver new drugs for common diseases [7]. Nevertheless, additional preclinical studies are certainly required to assess the potential of these anticancer drugs for their use in RIP-dependent diseases beyond cancer as well as potential toxicities, as RIPK3 has been reported to trigger apoptosis independently of its kinase activity [25]. Furthermore, it is necessary to take into consideration that Sorafenib may limit the anti-leukemic effects of cytotoxic drugs that induce necroptosis in leukemia cells. In support of this notion, Sorafenib has recently been shown to protect not only leukemia cell lines, but also primary, patient-derived leukemic blasts from Smac mimetic-induced necroptosis [12]. There is recent evidence showing that certain anticancer agents such as Smac mimetics alone or in combination with epigenetic drugs or glucocorticoids elicit necroptosis in acute leukemia cells when apoptosis is concomitantly blocked [27–29]. However, the question as to whether or not under certain conditions Sorafenib may alleviate the anti-leukemic activity remains to be addressed in additional models including in vivo models of leukemia.

In conclusion, repositioning FDA-approved anticancer drugs offers new perspectives for the development of drug-like inhibitors of necroptosis. Such inhibitors may be used for the treatment of clinical conditions and human diseases beyond cancer in which necroptotic cell death plays an important pathogenic role.

Funding Statement

This work has partially been supported by grants from the Bundesministerium für Bildung und Forschung (BMBF) (to S.F.).

Disclosure statement

The author declares that there is no conflict of interest.