Here we describe a pharmacological approach that we believe to be new to specifically bypass the apoptotic blockade to chemotherapy in multidrug-resistant ALL. Subcytotoxic concentrations of the BCL-2 antagonist obatoclax restored the response to dexamethasone by inducing a nonapoptotic cell death pathway. The activation of autophagy-dependent cell death in cells that are resistant to the apoptotic stimuli by dexamethasone is reminiscent of observations that were reported using experimental systems with defined genetic defects of key regulators of the intrinsic and extrinsic apoptotic response. In Bax–/–Bak–/–
MEFs and BCL-2–overexpressing MEFs, cell death triggered by etoposide or STS was dependent on autophagy genes beclin-1 and ATG-5 (11
). As also reported by others (6
), obatoclax was cytotoxic for Bax–/–Bak–/–
cell lines, which we demonstrate to be critically dependent on the autophagy pathway. Similarly, genetic or pharmacologic interference with caspase 8 or death receptor signalling can result in autophagy-dependent cell death with necroptotic features (12
). In lymphoid cells, this may constitute an alternative mechanism to control abnormal cellular proliferation in the absence of a normal apoptotic response. In activated T cells from mice with caspase-8 or FADD deficiency, autophagic signalling was required to induce RIP-1–dependent necroptotic cell death (41
). We here show that this mechanism of cell death can be activated in GC-resistant leukemia to restore the response to dexamethasone.
Autophagy has been recognized as an important regulatory mechanism of cell fate decisions. While it is clear that autophagy can have a protective function at times of cellular stress, the contribution of autophagy to the execution of programmed cell death is a subject of controversy (13
). Our data demonstrate that autophagic signalling is an integral part of the cell death mechanism when the response to dexamethasone is restored with obatoclax or rapamycin. Both inhibitors that interfere early (3-MA) or late (bafilomycin) with the autophagic process and knockdown of genes that are essential for autophagy, BECN1
, prevented resensitization to dexamethasone completely. Combination of dexamethasone and obatoclax inhibited clonogenic growth of GC-resistant ALL cells, and electron microscopy imaging unequivocally showed rapid induction of necrotic cell death with autophagic features. There is clear evidence that in cancer, autophagy is not necessarily a protective feature. For example, BECN1
was shown to act as a haploinsufficient tumor suppressor gene (42
), with increased frequency of spontaneous neoplasia, including lymphomas in beclin-1–haploinsufficient mice. The genes that are essential for the autophagic machinery are highly conserved and required in several autophagy-dependent cellular processes (44
). Several studies link autophagy genes to programmed cell death. In the central nervous system for example, ATG7 deficiency protected neurons from caspase-dependent and caspase-independent cell death after hypoxic/ischemic brain injury (45
). In human glioblastoma, knockdown of the autophagy genes ATG1
prevented the cytotoxic effect of cannabinoids, which induce autophagy-dependent cell death via an mTORC1-dependent pathway (46
). Autophagy was also shown to regulate programmed cell death during development. The steroid hormone ecdysone triggered autophagy-dependent cell death during morphogenesis of salivary glands from the larval stage to the adult stage in Drosophila
. This cell death pathway was independent of caspase activity (47
), providing compelling evidence for the modulation of an autophagic cell death pathway via steroid hormone signalling in normal development. Similarly, autophagy is required for programmed cell death in the midgut during Drosophila
metamorphosis, which provides additional evidence for specific regulation of cell death by autophagy, even in presence of an intact apoptotic machinery (48
). Combination treatment of obatoclax with GC drugs but not with other cytotoxic agents induced autophagy-dependent cell death in resistant ALL. This raises the question of whether autophagy is also required for the effect of dexamethasone in GC-sensitive ALL. A recent report described increased autophagy after dexamethasone treatment in GC-sensitive ALL cell lines, but cell death was associated with apoptotic features and knockdown of beclin-1 resulted only in partial rescue of this effect (49
). We also observed a partial reduction of dexamethasone cytotoxicity using 3-MA (Supplemental Figure 6) or nec-1 (Figure C) in a subset of GC-sensitive cell lines. We could, however, not detect an effect of 3-MA in primary ALL cells, in which caspase-dependent cell death prevailed. Our results identify autophagy as an early and limiting step to steroid sensitization by obatoclax in GC-resistant cells and underscore the importance of understanding the cellular context when designing strategies to target autophagy for cancer treatment.
There is clear evidence for hyperactivation of AKT (50
) and mTOR (5
) in GC-resistant ALL. Because mTOR is implicated in the control of autophagy in different settings (27
), hyperactive AKT-mTOR signalling could prevent induction of autophagy in resistant disease. We hypothesized that GC-resistant ALL cells could therefore be primed for mTOR-controled autophagy. Consistent with this idea, we found that induction of autophagic cell death by the combination of dexamethasone and obatoclax resulted in marked reduction in phosphorylation of the mTOR target S6 protein in resistant cells. The mechanisms by which obatoclax or rapamycin potentiate the effect of dexamethasone on mTOR appear to be different. Indeed combination of dexamethasone with rapamycin, but not with obatoclax, resulted in a marked decrease in phosphorylation of AKT on Ser473, consistent with recent finding showing that mTOR can also act upstream of AKT (51
). Modulation of mTOR target phosphorylation was only seen when obatoclax was combined with dexamethasone, but not with other cytotoxic agents, suggesting that dexamethasone exposure contributes to inhibition of mTOR. Indeed, exposure to dexamethasone was reported to be associated with repression of mTOR signalling in myoblast cell lines and lymphoid cells (28
). The importance of mTOR for the control of autophagy is also underscored by the results of a comprehensive screen using a chemical compound library in order to identify new pharmacologic inducers of autophagy, in which proautophagic activity of candidate molecules was always associated with decreased phosphorylation of mTOR targets (53
). Extensive studies will be required to dissect the primary signalling events triggered by the combination of obatoclax and dexamethasone, as they may provide important clues about the mechanisms of drug resistance in ALL.
GC resistance does not appear to be associated with genetic or functional defects of the GC receptor in ALL (54
). Dexamethasone was proposed to induce apoptosis by increasing the levels of the BH3-only proapoptotic protein BIM, which were markedly reduced in selected cases of GC-resistant ALL (32
). However, in most cases tested, we could not detect induction of BIM by dexamethasone in GC-sensitive cell lines and primary ALL cells. Furthermore, we did not detect increased levels of BIM, decreased MCL-1 protein levels, or increased MCL-1 protein turnover associated with steroid sensitization. Instead, we found, by immunoprecipitation of endogenous proteins, subcytoxic concentrations of obatoclax to result in the disruption of MCL-1 with beclin-1 in ALL cells. This suggests the possibility that MCL-1 could control induction of autophagy via beclin-1, as it was proposed for other BCL-2 family members recently (14
). Interestingly, this effect was not seen in cells treated with rapamycin, again pointing out that the target mechanism of obatoclax is different. We recognize the fact that this effect could also result from indirect mechanisms on the protein complex, including MCL-1 and beclin-1. Furthermore, functional experiments modulating MCL-1 expression levels were not conclusive. Knockdown of MCL-1 resulted in moderate activation of apoptosis and partial sensitization to dexamethasone that was caspase dependent but prevented complete resensitization to dexamethasone by obatoclax and autophagic cell death. Activation of apoptosis is possibly overriding induction of autophagy in this context. In support of this hypothesis, we detected activation of caspase-9 after downregulation of MCL-1, with caspase-9–dependent cleavage of beclin-1. This provides a possible mechanism to prevent autophagy induction when apoptosis is activated. Taken together, steroid modulation with low-dose obatoclax did not involve release of proapoptotic BCL-2 family proteins from MCL-1 but triggered autophagy-dependent cell death by a mechanism that required the presence of MCL-1.
In this context, activation of autophagy is required to induce necroptosis, a form of programmed necrosis that has been described to occur when apoptosis is abortive due to caspase inhibition (16
). Execution of necroptosis is dependent on the RIP-1 kinase (17
). Our data demonstrate that RIP-1 activity is absolutely required for steroid sensitization by obatoclax and by rapamycin. The cell death morphology that was documented by electron microscopy is consistent with the morphology reported for necroptosis (12
). Furthermore, the deubiquitinase cylindromatosis (CYLD), which has been shown to regulate RIP-1 (38
), was functionally required for execution of necroptosis. This is consistent with experiments showing that RIP-1 kinase activity was required to trigger cell death and that ubiquitination of RIP-1 prevents cell death signalling (16
). RIP-1 and CYLD were included among the core genes that were identified in a functional siRNA screen for genes that were essential for necroptosis (18
). To the best of our knowledge, we provide the first clinically relevant model with consistent validation in primary leukemia cells from patients with very high risk disease, in which the necroptotic pathway can be exploited to restore response to therapy. As such, this will constitute a very relevant experimental model to study the mechanisms of necroptosis in depth. Our results clearly imply a direct link between the autophagic pathway and RIP-1–mediated signalling events leading to necroptosis. Autophagy is triggered early and independent of RIP-1 kinase activity, indicating that it acts upstream of necroptotic signals. A number of studies identify RIP functionally as part of a complex with proteins of the death receptor pathway, such as FADD and caspase-8 (35
). Experiments using mouse models indicate that autophagy and necroptosis could be linked via recruitment of components of the death receptor pathway to the membrane of autophagosomes (41
). It is tempting to speculate that a similar mechanism is triggered in ALL cells upon costimulation with dexamethasone and obatoclax. Our findings warrant extensive biochemical follow-up studies to understand how RIP-1 is activated and how the autophagic machinery is connected to the necroptotic pathway.
Based on promising studies by others (32
), we have established a leukemia xenograft model of de novo highly resistant ALL. The power of this approach resides in the possibility to select cases from relevant patient groups, starting from cryopreserved leftover diagnostic samples, from one of the largest cooperative trials for the treatment of childhood ALL. By focusing on cases with VHR-ALL by MRD, we also selected for patients that are most significantly resistant to prednisone in vivo, as defined by the reduction of leukemia cells in the peripheral blood after 1 week of prednisone monotherapy. Accordingly, ALL cells from these patients were completely resistant to dexamethasone and other chemotherapeutic agents in vitro. Low-dose obatoclax restored the response to dexamethasone, both in precursor B cell and T cell ALL cases. The durable remissions observed with 1 year follow-up, using the leukemia xenograft model, are indicative of strong antileukemic activity. Furthermore, the broad chemosensitizing effect of low-dose obatoclax in combination with daunorubicin, vincristine, and cytarabine in multidrug-resistant primary ALL cells provides a strong basis for further evaluation of obatoclax in combination with a multidrug regimen. The xenograft approach will be essential to validate this approach for heavily pretreated relapse and refractory patients. Our observation that isolated clones can emerge in clonogenic assays after treatment of a cell line with obatoclax and dexamethasone indicates that resistance to this approach may occur. Our established xenograft system will enable us to screen a larger number of ALL cases to verify whether resistance to this approach has to be expected. The identification of resistant cases would provide a model to establish markers that correlate with response or resistance. Based on current data, dynamic changes of mTOR activity with treatment represent a good candidate marker. Such knowledge will serve to optimize patient selection for clinical trials.
Taken together, our data support a model in which the apoptotic blockade in GC-resistant ALL cells can be overcome by activating an autophagy-dependent necroptotic cell death pathway. The characteristic necroptotic features by electron microscopy and the changes in the phosphorylation profile of S6 protein provide tools to assess the biological response to combination treatment with obatoclax and dexamethasone in patients in refractory ALL. Given the acceptable toxicity profile of obatoclax in clinical studies in adults with hematologic malignancies (9
), our study provides a compelling rationale for the evaluation of this new pharmacological strategy for the treatment of children with refractory and relapsed ALL.