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Targeting sphingolipid metabolism as an approach for combination therapies in haematological malignancies

Targeting sphingolipid metabolism as an approach for combination therapies in haematological... Conventional chemotherapy-based drug combinations have, until recently, been the backbone of most therapeutic strategies for cancer. In a time of emerging rationale drug development, targeted therapies are beginning to be added to traditional chemotherapeutics to synergistically enhance clinical responses. Of note, the importance of pro- apoptotic ceramide in mediating the anti-cancer effects of these therapies is becoming more apparent. Furthermore, reduced cellular ceramide in favour of pro-survival sphingolipids correlates with tumorigenesis and most importantly, drug resistance. Thus, agents that manipulate sphingolipid metabolism have been explored as potential anti-cancer agents and have recently demonstrated exciting potential to augment the efficacy of anti-cancer therapeutics. This review examines the biology underpinning these observations and the potential use of sphingolipid manipulating agents in the context of existing and emerging therapies for haematological malignancies. Facts � Efficacy of many chemotherapeutics and targeted therapies is dictated by cellular ceramide levels. � Oncogene activation skews sphingolipid metabolism to favour the production of pro-survival sphingolipids. � Inhibitors of enzymes involved in ceramide metabolism exhibit promise in the relapsed-refractory setting. � Anti-cancer activity of sphingosine kinase inhibitors provides several options for new drug combinations. Open Questions � What other clinically utilised drugs rely on increases in ceramide levels for their efficacy and can they be effectively partnered with other ceramide inducing agents? � How does ceramide modulate the Bcl-2 family proteins, Mcl-1 and Bcl-2? � Are sphingolipid enzyme inhibitors best suited in the frontline or relapsed-refractory setting? Introduction undergone little change over the last 30 years. The success Frontline chemotherapeutic regimens for the majority of tyrosine-kinase inhibitors (TKI) in chronic myeloid of haematological malignancies have, until recently, leukaemia (CML) has significantly increased the 10-year survival rate to 83%, enabling some patients to cease therapy (~40%) and achieve long-term remission Correspondence: Stuart M. Pitson (stuart.pitson@unisa.edu.au) (~40% >3 years) . However, unlike CML which is driven Centre for Cancer Biology, University of South Australia and SA Pathology, UniSA CRI Building, North Terrace, Adelaide, SA 5001, Australia solely by the BCR-ABL oncogene, most blood cancers are Adelaide Medical School, University of Adelaide, Adelaide, SA 5000, Australia more genetically heterogeneous displaying complex clonal Full list of author information is available at the end of the article. These authors contributed equally: Jason A. Powell, Stuart M. Pitson Edited by: A. Rufini © The Author(s) 2018. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Lewis et al. Cell Death Discovery (2019) 5:4 Page 2 of 11 Fig. 1 Overview of the sphingolipid cycle. The pleotropic nature of ceramide allows a promotion of multiple cellular fates including survival, migration and angiogenesis. Furthermore this also prevents a lethal accumulation of apoptotic sphingolipids (red) such as ceramide by maintaining a balance of pro-survival lipids (blue). Due to the propensity of transformed cells to deplete ceramide by increasing expression of enzymes, such as SPHK1/2 and GCS, inhibitors targeting these enzymes have exhibited therapeutic potential by tipping the balance to favour ceramide accumulation and promote cell death. architecture with conventional chemotherapeutic agents palmitoyl-CoA by the rate-determining enzyme, serine remaining the backbone of most therapy regimens. One palmitoyltransferase to 3-keto-dihydrosphingosine mechanism whereby chemotherapy induces apoptosis in (Fig. 1) . Following further modification to dihydro- malignant cells is through increases in the cellular levels sphingosine, ceramide synthases convert dihydro- 2,3 of the pro-apoptotic sphingolipid, ceramide . Sphingo- sphingosine to dihydroceramide which are then lipids are a class of lipids that can exert pleotropic cell desaturated to generate ceramides . From here, these signalling effects. Ceramide is a central component of ceramides can be modified to various sphingolipid species sphingolipid metabolism that is tightly regulated due to its that modulate membrane composition and signal trans- pro-apoptotic effects (Fig. 1). Enzymes involved in the duction. For example, ceramides can be glycosylated by conversion of ceramide to other sphingolipids have been glucosylceramide synthase to glucosylceramides which implicated in drug resistance by depleting ceramide to can serve as an intermediary for other glycosphingolipids, produce pro-survival sphingolipids (Table 1). Indeed, phosphorylated by ceramide kinase, modified by the inhibitors targeting these enzymes have been shown to addition of phosphocholine by sphingomyelinase (SMase) induce cell death through inducing the accumulation of to form sphingomyelins, or deacylated by ceramidases to 4–7 lethal levels of ceramide . Thus, manipulating sphin- form sphingosine and subsequently, through the action of golipid metabolism has shown promise in combination sphingosine kinases (SPHKs), generate sphingosine-1- with conventional chemotherapy, as well as with novel phosphate (S1P) (Fig. 1) . Notably, considerable evidence agents. In this review, we highlight the literature that implicates important roles for several of these sphingoli- examines how targeting sphingolipid metabolism shows pid metabolic enzymes in tumorigenesis and resistance to considerable promise for chemo-sensitising patients with therapy in haematological malignancies (Table 1). blood cancers. Sphingosine kinases The sphingolipid cycle The “sphingolipid rheostat” is a concept that describes While originally assumed to just play an integral role in cell fate as a balance between pro-apoptotic ceramide and 9,10 the structure of the cell membrane, sphingolipids have pro-survival S1P . As the SPHKs are critical in the only been found to be prominent players in cell signalling, exit point for degradation of sphingolipids, via conversion capable of exerting a myriad of cell responses. De of sphingosine to S1P, and then its degradation by S1P novo sphingolipid production commences at the endo- lyase, these enzymes represent one of the key players in plasmic reticulum with the condensation of serine and maintaining ceramide levels (Fig. 1). Furthermore, S1P Official journal of the Cell Death Differentiation Association Lewis et al. Cell Death Discovery (2019) 5:4 Page 3 of 11 Table 1 Role of sphingolipid enzymes in haematological Activated SPHK1 subsequently translocates to the plasma malignancies membrane, via an interaction with calcium and integrin binding protein 1 (CIB1), to convert sphingosine to Enzyme Malignancy Role 12–14 S1P . Due to the frequency with which hyperactiva- tion of the RAS pathway occurs in cancer, constitutive Acid ceramidase AML Increased expression in patient samples. phosphorylation and activation of SPHK1 in cancer is Modulates Mcl-1 expression in a post- likely to be common, promoting drug resistance by not translational manner only depleting ceramide levels but also promoting pro- Ceramide AML Suppressed by FLT3 signalling. Mediates 9,15,16 survival signalling . Furthermore, drug resistant cell synthase cytotoxicity of FLT3 inhibitors by lines have been shown to also exhibit increased SPHK1 induction of lethal mitophagy activity and attenuate increases in ceramide which would 15–17 Glucosylceramide AML Overexpressed in chemotherapy otherwise promote apoptosis . Several studies have 29,30 synthase resistance cell lines shown that targeting SPHK1 can resensitise cells to che- CLL Upregulated in response to B-cell motherapy, by blocking the conversion of sphingosine to 15, receptor stimulation S1P and generating a bolus of pro-apoptotic ceramide 16,18 98 . Lymphoma Potential role in tumour initiation Despite catalysing the same reaction, characterising Myeloma Potential role in tumour initiation SPHK2 has proved more elusive with conflicting literature 19,20 Sphingosine AML Overexpressed in patient samples. on its role in tumorigenesis . Nevertheless, targeting kinase 1 Increases drug resistance to SPHK2 has shown promise in a number of malignancies, chemotherapy and ceramide inducing including breast cancer , acute lymphoblastic leukaemia 16,18 22 5,23 strategies (ALL) and myeloma . ALL Overexpressed in patient samples Ceramidase CML Overexpressed in Imatinib-resistant cell Breakdown of ceramide to sphingosine is mediated by lines . Upregulates Mcl-1 in a BCR-ABL ceramidases, with several homologues described that dependent manner . Represses PP2A to function in acidic, neutral, or alkaline pH. The most promote BCR-ABL stability characterised form, acid ceramidase (AC) is thought to Sphingosine ALL Promotes B-ALL disease progression. localise to acidic compartments, such as lysosomes . The kinase 2 Inhibits histone deacetylases to promote degradation of ceramide by AC has highlighted a potential Myc expression mechanism for drug resistance by degrading the bolus of Myeloma Upregulated in cell lines and patient ceramide induced by cancer treatments. Overexpression 5,23 samples of AC has been reported in several solid tumours in addition to acute myeloid leukaemia (AML) . Pre-clinical AML acute myeloid leukaemia, B-ALL B cell acute lymphoblastic leukaemia, BCR- ABL Breakpoint cluster region–Abelson murine leukaemia viral oncogene studies utilising AC inhibitors have shown efficacy in homolog 1, CLL chronic lymphocytic leukaemia, FLT3 FMS-like tyrosine kinase 26,27 resensitising cells to chemotherapeutics . Although no 3, PP2A protein phosphatase 2A direct AC inhibitors are currently under investigation in clinical trials, interestingly, the clinically available oes- generation by SPHK also directly promotes cell survival as trogen receptor antagonist, Tamoxifen has demonstrated well as activating oncogenic signalling pathways by acting AC inhibition, warranting potential investigation as an as both an intracellular second messenger, and as a ligand adjuvant for chemotherapy regimens . for a family of five S1P-selective G-protein-coupled receptors . Glucosylceramide synthase Although very similar enzymes, some sequence and Glucosylceramide synthesis is the first step in the gen- presumed structural diversity between the two human eration of complex glycosphingolipids (Fig. 1). Much of SPHKs, SPHK1 and SPHK2, is thought to drive partially the interest in glucosylceramide has focussed on its role as different biological functions of these enzymes .Itis a “sink” for ceramide, with the action of glucosylceramide generally accepted that SPHK1 is associated with a pro- synthase (GCS) shunting ceramide through glucosylcer- tumorigenic role. Under homeostatic conditions, basal amide and the glycosphingolipid pathway. It is no surprise SPHK1 activity is thought to maintain ceramide levels that the removal of ceramide through this pathway in the absence of stimuli to prevent inappropriate has been proposed as a mechanism of drug resistance cell death . Activation of SPHK1 can occur via the and multiple studies have identified GCS as a therapeutic RAS pathway with extracellular signal-regulated kinases target by preventing the accumulation of lethal levels 12 29–32 1/2 (ERK1/2) phosphorylating SPHK1 at Serine 225 . of ceramide . The approval of the GCS inhibitor, Official journal of the Cell Death Differentiation Association Lewis et al. Cell Death Discovery (2019) 5:4 Page 4 of 11 Eliglustat, for clinical use in the lysosomal storage dis- inducing factor and second mitochondria-derived acti- order, Gaucher’s disease, suggests that GCS inhibition is vator of caspases (SMAC) . well tolerated in humans and could provide a means to investigate combinational studies of GCS inhibition with Targeting perturbations in sphingolipid chemotherapeutics and targeted agents. metabolism in haematological malignancies Clearly, sphingolipid metabolism is frequently dysre- Ceramide synthase gulated in haematological malignancies, and can confer In mammals, six different ceramide synthases (CerS1-6) resistance to many drugs currently employed to treat have been recognized, each capable of generating varying these diseases. Thus, there appears potential therapeutic ceramide species of differing fatty acyl chain lengths in benefit in combining sphingolipid modulators with clin- both the de novo sphingolipid pathway as well as the ical chemotherapeutics and novel therapies for the treat- conversion of sphingosine to ceramides in the so-called ment of a range of blood cancers. sphingolipid salvage pathway (Fig. 1) . Several studies have demonstrated that CerS activity is crucial to the Chronic myeloid leukaemia cancer cell killing efficacy of chemotherapeutics , The BCR-ABL inhibitor Imatinib and subsequent TKIs, tumour necrosis factor-related apoptosis-inducing ligand Dasatinib and Nilotinib have dramatically improved sur- 36 37 (TRAIL)-induced apoptosis , radiation and kinase vival rates for CML, with some patients even dis- 38,39 inhibitors . Although activation of CerS presents an continuing treatment due to prolonged molecular 55,56 attractive therapeutic target, CerS regulation remains remission status . The emergence of ATP binding site poorly understood . mutations within BCR-ABL, such as the T315I mutant, however, confers resistance to most of these ATP- Cellular ceramides are increased by chemotherapy competitive inhibitors, and presents a key therapeutic Chemotherapy frequently promotes the accumulation issue . The recent development of allosteric BCR-ABL of ceramide, which appears to contribute to induction of inhibitors, GNF-2 and ABL001 may provide an alternative 41–43 58 cancer cell death . While the mechanisms responsible option in the future to prevent this . Analysis of GNF-2 for this induction of ceramide depends on the che- treated cells revealed an increase in ceramide levels sug- motherapy employed, numerous studies have observed gesting BCR-ABL may suppress ceramide synthesis . chemotherapy-induced activation of CerS and acid Augmentation of ceramide levels using the glucosylcer- 41–44 SMase, as well as inhibition of AC amide synthase inhibitor D-threo-l-phenyl-2-decanoyla- . Notably, among the genes activated by p53 in response to chemotherapy, mino-3-morpholino-1-propanol (PDMP), enhanced 37 45 46 CerS5 , CerS6 and neutral SMase are prominent. apoptosis and resensitised T315I mutant CML cells to There is also evidence to suggest that the cysteine pro- both Imatinib and Nilotinib suggesting co-targeting tease, Cathepsin B can degrade SK1 following p53 upre- BCR-ABL with sphingolipid modulating agents may be a gulation by genotoxic stress, increasing ceramide levels . novel strategy to further enhance the efficacy of TKI Thus, with the widespread effect of chemotherapy treatment. and p53 on manipulating sphingolipid metabolism, direct Comparative analysis of Imatinib sensitive and resistant modulation of sphingolipid enzymes in combination K562 CML cells revealed enhanced S1P generation and with current agents may provide improved clinical reduced ceramide levels as a consequence of SPHK1 outcomes. upregulation compared to parental Imatinib sensitive K562 cells , suggesting a potential role for SPHK1 in Mechanisms of ceramide-induced cell death Imatinib resistance. Notably, overexpression of SPHK1 Ceramide exerts its tumour suppressive activities blocked the cytotoxic effects of Imatinib in sensitive K562 through multiple mechanisms, including activation of cells recapitulating the phenotype of Imatinib resistance . protein phosphatases 1 (PP1) and 2A (PP2A) , and Findings by Li et al. corroborated this relationship with 49 50 suppression of oncogenes such as Akt , c-Myc and BCR-ABL upregulating SPHK1 activity through MAPK, Bcr–Abl . Perturbation of sphingolipid homeostasis and PI3K and JAK2 signalling suggesting a positive feedback ceramide accumulation within cell membranes has loop . Genetic and chemical targeting of SPHK1 also also been reported to invoke pro-apoptotic signalling uncovered a positive relationship between SPHK1 and through activation of the unfolded protein response , anti-apoptotic protein Mcl-1, whereby overexpression of 52 39,53 autophagy and mitophagy . Furthermore, there is SPHK1 increased Mcl-1 levels in a BCR-ABL dependent strong evidence suggesting that ceramide can directly manner . initiate apoptosis by the formation of channels within the The precise mechanism between SPHK1 and BCR- mitochondrial outer membrane, capable of facilitating ABL stability was later uncovered with S1P receptor 2 the release of proteins, such as cytochrome c, apoptosis- (S1P ) supressing PP2A-mediated de-phosphorylation Official journal of the Cell Death Differentiation Association Lewis et al. Cell Death Discovery (2019) 5:4 Page 5 of 11 51 68 and subsequent degradation of BCR-ABL . Targeting kinase (BTK) activity . Notably, treatment of CLL cells S1P/S1P signalling resensitised Imatinib-resistant K562 with the PI3Kδ inhibitor, Idelalisib or BTK inhibitor, 51 69 cells and T315I patient blasts to BCR-ABL inhibition . Ibrutinib which are currently FDA approved for CLL , Notably, elevated SPHK1 and S1P mRNA levels were abrogated increases in GCS transcription and synergised found in T315I patient blasts used in this study which with the first generation Bcl-2/Bcl-xl inhibitor ABT- 7,67,68 raises the question as to whether there is a correlation 737 . These data suggest that combining ceramide- between these genes and BCR-ABL mutational status . inducing strategies with Bcl-2 inhibitors may exhibit BCR-ABL inhibitors, Dasatinib, Nilotinib and GFN-2 synergistic activity in CLL. have also been shown to induce transcription of various A recent study by Dielschneider et al. investigating the 59,61,62 CerS genes to upregulate ceramide production . use of “lysosomal penetrating” or lysosomotropic agents Intriguingly, Dasatinib treatment was associated with in CLL uncovered an increase in S1P phosphatase 1 increases in CerS2,5,6 whereas Nilotinib was associated (SPP1) and sphingosine in primary CLL patient samples 61,62 with increases in Cers5 expression . Combining either compared to normal B cells. Lysomotropic agents accu- Dasatinib or Nilotinib with the GCS inhibitor, PDMP, mulate within lysosomes, promoting the release of lyso- augmented apoptosis, presumably through synergistic somal contents such as cathepsins and the initiation 61 71 increases in ceramide levels . The interplay between of non-apoptotic cell death . As a natural detergent BCR-ABL, SPHK1 and CerS highlights the oncogenic and lysomotropic agent, sphingosine has been shown potential of BCR-ABL in skewing sphingolipid metabo- to induce lysosome destabilisation and thus may lism to favour a pro-survival phenotype, and highlights a explain the susceptibility of CLL cells to lysomotropic potential therapeutic benefit for combining ceramide agents . The addition of either sphingosine or SKI- inducers with BCR-ABL inhibitors. II augmented the apoptotic response of the lysosomo- tropic agent Siramesine, against primary CLL cells . The Chronic lymphocytic leukaemia clinical relevance of the above findings by Dielschneider Chronic lymphocytic leukaemia (CLL) is an indolent et al. are intriguing particularly with reports of the anti- form of leukaemia with some patients not requiring CD20 antibody Obinutuzumab inducing lysosomal per- treatment in their lifetime . CLL patients with unfa- meabilisation in B-cell malignancies, highlighting its vourable genetic factors such as 17p deletion (del(17p)) potential use in combination with ceramide/sphingosine- however respond poorly to frontline therapy in part due generating agents . to the absence of p53 . The addition of the Bcl-2 inhi- bitor, Venetoclax has been a paradigm shift for p53 null Acute lymphoblastic leukaemia CLL patients with 79% of relapsed/refractory patients in a There is disparity in treatment outcome for ALL with phase II clinical trial responding, 20% of whom exhibited cure rates for adults at 30–40% despite paediatric cases 65 74 undetectable disease by flow cytometry . As always, being closer to 90% . Poor risk subtypes such as Phila- resistance to drugs remains a problem with other Bcl-2 delphia (Ph)-like and Ph positive ALL are frequently family members such as Mcl-1, identified as a marker of observed in adults (~50%) and typically respond poorly to 66 75 resistance to Venetoclax . Although still effective in CLL chemotherapy . The addition of a TKI to standard che- patients, identifying other drugs to use alongside Vene- motherapy for Ph positive patients has improved treat- toclax and prevent the emergence of resistance is crucial. ment response and survival rates for many of these In the context of sphingolipid metabolism, the Bcl-2/ patients with initial studies showing complete haemato- Bcl-xl inhibitor Navitoclax was found to enhance CerS logical response in ~90% of patients . Like CML, activity and increase C16 ceramide synthesis. Although, relapsed Ph positive ALL patients exhibit similar muta- the mechanism remains to be confirmed, the authors to tions in BCR-ABL that impart TKI resistance, requiring a proposed that Bcl-2 inhibition allows Bak to interact with move to second and third generation BCR-ABL inhibitors. CerS5 or 6 resulting in the production of C16 ceramide . A potential role for the SPHKs in ALL was first high- Furthermore, combining Navitoclax with either the GCS lighted using studies with the pan SPHK inhibitor, SKI-II, inhibitor, PDMP, or the SPHK inhibitor, SKI-II-induced in combination with the commonly used chemother- 7 77 synergistic increases in ceramide and cell death . apeutic Vincristine . SKI-II treatment induced cell death Interestingly, B-cell receptor (BCR) signalling in pri- in ALL cell lines and primary lymphoblasts. As expected, mary CLL cells has been shown to induce glucosylcer- inhibition of the SPHKs and the accompanying increase in amide generation, potentially blunting the efficacy of ceramide synergised with Vincristine treatment . How- Rituximab treatment . Schwamb et al. proposed that ever, combining the SPHK2 inhibitor, ABC294640 with BCR signalling stimulates GCS transcription upon IgM Doxorubicin or Vincristine elicited only an additive effect treatment of primary CLL cells in a manner dependent on suggesting the synergy observed may be a consequence of phosphoinositide 3-kinase (PI3K)δ and Bruton’s tyrosine SPHK1 inhibition . Official journal of the Cell Death Differentiation Association Lewis et al. Cell Death Discovery (2019) 5:4 Page 6 of 11 A role for SPHK1 in contributing to the development of dependent cell death implicating mitochondrial mediated BCR-ABL driven ALL has been described. Gene expres- apoptosis . Although, LCL-204 appeared to display some sion data from two separate patient cohorts revealed a anti-leukaemic activity in patient-derived xenografts significant increase in SPHK1 expression in BCR-ABL in vivo, the assessment of circulating blasts within the positive ALL compared with BCR-ABL negative cases, periphery as opposed to bone marrow makes it difficult to highlighting a potential relationship that had previously draw conclusions from these studies. Overexpression of 17,60 been described in CML . Deletion of SPHK1 in BCR- AC correlated with an increase in Mcl-1 expression and ABL positive, but not BCR-ABL negative murine ALL blunted the cytotoxic effects of Bcl-2/Bcl-X inhibitor, models delayed disease incidence implicating SPHK1 as a ABT-737. Whilst Tan et al. demonstrate that AC inhibi- significant player in BCR-ABL driven ALL . Further- tion results in proteasome-mediated degradation of Mcl- more, combining SPHK inhibitors, SK1-I, SKI-II or 1, the exact mechanism of how AC upregulates ABC294640 with Imatinib also induced synergistic cell Mcl-1 was not further explored. Thus, the targeting of death in BCR-ABL positive cell lines which collectively Mcl-1 through AC inhibition warrants further investiga- warrants further investigation . tion in combination with Bcl-2 inhibitor, Venetoclax. Enhanced SPHK2 protein levels and activity in ALL Chemoresistant AML cells were also susceptible to cell patient samples and cell lines when compared with nor- death induced by AC inhibition which was correlated with 22 4 mal B-cell progenitors has also been reported . SPHK2 the increase in cellular ceramide levels . Although pre- inhibition was associated with reductions in histone liminary, these findings serve as a basis for further acetylation of the c-Myc promoter representing new evi- investigating the potential for AC inhibitors as chemo- dence for an oncogenic role for SPHK2 . Reductions resensitisers in the relapse/refractory setting. in c-Myc expression were observed in BCR-ABL The oestrogen receptor antagonist, Tamoxifen has also transformed B-ALL cells from SPHK2 knockout mice, demonstrated potent inhibition (IC ~1 μM) of GCS and translating into increased overall survival in vivo . These has demonstrated anti-leukaemic activity. Work by pre-clinical studies may provide the impetus to assess Morad et al. utilising short chain ceramides in combina- the addition of ABC294640 alongside BCR-ABL inhibitors tion with Tamoxifen revealed synergistic cell death in in patients with Ph positive ALL. AML cell lines as a consequence of mitochondrial meta- bolic collapse typified by decreases in ATP levels and Acute myeloid leukaemia glycolytic flux . Tamoxifen treatment prevented the Among drugs in the development pipeline for AML, accumulation of glucosylceramide species, in agreeance selective FLT3 inhibitors Crenolanib and ASP2215 have with the proposed role of these molecules as a “sink” for exhibited impressive single-agent activity (ORR 52%, CR excess cellular ceramide . Besides the demonstrated 41%) in a phase I/II study of relapsed/refractory AML . effects on GCS, Tamoxifen has also been reported to Recently, work by Dany et al. examined the mechanism of inhibit AC which may contribute to its cytotoxic effects . action of FLT3 inhibitors, including Crenolanib, and Nethertheless, the off-target inhibition of GCS by observed reductions in C18-ceramide levels in FLT3 Tamoxifen, presents an interesting therapeutic angle positive AML patient blasts . The authors attributed this given its approval for hormone-dependent breast cancer, to striking reduction in CerS1 mRNA levels, suggesting an potentially allowing fast track approval as adjuvant ther- inverse relationship between FLT3 activity and CerS1 apy in combination with chemotherapy. expression. Restoration of CerS1 upon treatment with Confirmation of SPHK1 as a therapeutic target in AML FLT3 inhibition resulted in mitophagy-dependent cell has been established by several groups using both genetic death suggesting repression of ceramide synthesis may be knockdown of SPHK1 and chemical SPHK1 inhibitors. an important step in AML pathogenesis. Intriguingly, a Chemotherapy resistant AML cell lines exhibited a lack of mitochondrial targeted ceramide analogue effectively ceramide generation upon drug treatment suggesting an suppressed FLT3 inhibitor resistant AML patient samples involvement of SPHK1 as a mediator of drug resistance . in vivo suggesting the reactivation of mitochondrial Overexpression of SPHK1 in chemo-sensitive AML cell ceramide synthesis downstream of FLT3 signalling may lines imparted resistance to chemotherapeutics confirming 16,18 be beneficial in overriding FLT3 resistance. SPHK1 as a marker of drug resistance in AML . Genetic Several groups have observed changes in AC in solid and chemical inhibition of SPHK1 in AML cell lines and 26,27,80 84 tumours , with its role in AML recently described. primary patient samples induced cell death and synergised Upregulation of AC mRNA and activity was observed in with chemotherapeutic agents . Interestingly, SPHK1 AML patient samples compared with normal CD34 inhibition-induced synergistic cell death with cytarabine in 4 18 bone marrow cells . Treatment of AML cell lines with the the refractory leukaemic initiating cell (LIC) population . AC inhibitor LCL-204 was associated with rapid loss of Although these synergistic effects require further in vivo the pro-survival Bcl-2 family protein, Mcl-1 and caspase evaluation, the findings suggest addition of an SPHK1 Official journal of the Cell Death Differentiation Association Lewis et al. Cell Death Discovery (2019) 5:4 Page 7 of 11 Fig. 2 Targeting sphingolipid metabolism in AML. An overview of targeting sphingolipid enzymes (red) in combination with pre-clinical or clinically utilised drugs (blue) in AML. inhibitor to a standard chemotherapeutic regimen has the expression, as well as the synergy observed with Bcl-2 7,18 potential to greatly enhance clinical responses and reduce inhibitors , provides considerable impetus to further relapse rates by targeting the LIC population. explore how ceramide modulates Mcl-1 stability. Recently, a link between SPHK1 and Mcl-1 was eluci- Targeting of S1P in AML has shown to induce loss-of- dated by Powell et al. whereby SPHK1 inhibition resulted in Mcl-1 and synergised with Bcl-2 inhibition recapitulating 18 18 Mcl-1 degradation . Loss-of-Mcl-1 coincided with induc- the observations with SPHK1 inhibition . A link between tion of BH3-only proteins, particularly Noxa, a known S1P and Mcl-1 stability has been largely overlooked in inducer of Mcl-1 degradation . As Mcl-1 is a marker of the context of cancer biology. We speculate that a change resistance to Bcl-2 inhibitor, Venetoclax, this link highlights in Mcl-1 phosphorylation may be responsible for loss-of- a new angle to target Mcl-1 and enhance the efficacy of Mcl-1 associated with S1P inhibition highlighting Venetoclax which is exhibiting impressive single-agent potential involvement of PP2A based on findings observed activity (ORR 79%) in CLL, a malignancy highly depen- in CML . Furthermore, this suggests that the loss-of- dent on Bcl-2 but only exhibited modest single-agent SPHK1 and its effect on Mcl-1 is two-pronged with activity in AML (ORR 19%) . Follow-up trials with Vene- reduced S1P/S1P signalling and the accumulation of toclax in combination with chemotherapy and hypo- ceramide due to the lack of sphingosine processing by methylating agents are currently ongoing with initial reports SPHK1. Despite the lack of targeted therapies in wide- 87,88 showing promising results . Pre-clinical evidence com- spread clinical use, the potentially broad applicability of bining SPHK1 and Bcl-2 inhibition showed synergistic cell inhibitors of sphingolipid metabolism in AML may pro- death in AML cell lines by targeting both Mcl-1 and Bcl-2 . vide significant benefit in a malignancy whose treatment Although, the exact mechanism is currently unknown, the options are reliant on ceramide accumulation for their 4 18 work from both Tan et al. andPowelletal. , suggests efficacy (Fig. 2). ceramide and S1P regulate Mcl-1 degradation. A link between ceramide and resistance to Bcl-2 tar- Multiple myeloma geting strategies has previously been identified in small The repertoire of novel therapies to treat multiple cell lung carcinoma which could explain the synergy myeloma has markedly increased over the past two dec- between the two drugs . Gene correlation analysis for ades. Of these novel therapies, the proteasome inhibitors Navitoclax resistance, identified an atypical Bcl-2 protein, (PIs), particularly Bortezomib (Velcade), have resulted in a Bcl-rambo, as a direct inhibitor of CerS2 and −6 . This marked improvement in overall response and median finding supports in vitro studies demonstrating that the survival rates (from 3 to 6–7 years) . Sustained mono- GCS inhibitor, PDMP synergises with Navitoclax sug- clonal immunoglobulin production by myeloma plasma gesting ceramide augments Bcl-2 targeting strategies . cells induces endoplasmic reticulum (ER) stress and The ability of sphingolipid modulating agents such as activates the unfolded protein response (UPR) which aims 4 18 those directed against AC or SPHK1 to reduce Mcl-1 to reduce global protein translation and correct the Official journal of the Cell Death Differentiation Association Lewis et al. Cell Death Discovery (2019) 5:4 Page 8 of 11 Fig. 3 Exploiting sphingolipid synthesis to enhance the efficacy of proteasome inhibitors. Due to the constitutive production of immunoglobulin by malignant plasma cells, their reliance on the unfolded protein response (UPR) to prevent an accumulation of misfolded proteins with the endoplasmic reticulum (ER) for survival renders them susceptible to inducers of ER stress. As the site of de novo sphingolipid synthesis, accumulation of saturated lipids such as ceramide within the ER, induces a lipid dependent UPR, promoting apoptosis. 91 23 misfolded protein-induced stress placed on the ER .To upregulation , just as it has been shown for Bortezo- effect this, proteins that cannot be correctly folded are mib , providing evidence for combination therapies. As transported to the 26S proteasome for degradation, expected, the loss-of-Mcl-1 observed with ABC294640 highlighting the relevance of PIs, which block proteasome treatment sensitised myeloma cells to Venetoclax . activity and commit the myeloma cell to apoptosis due to Indeed, combination of the SPHK2 inhibitor K145 and the accumulation of misfolded proteins in the ER lumen Bortezomib has been shown to result in synergistic anti- 92 5 and activation of a terminal UPR . myeloma effects in vitro and in vivo . These findings are Unlike other haematological malignancies, SPHK2 supported by studies showing that changes in lipid appears to be the dominant SPHK isoform in mye- saturation in the ER membrane, potentially including the 5,23 loma . Both pharmacological inhibition of SPHK2 using accumulation of sphingolipids, can activate the UPR ABC294640 or K145, and genetic interference of SPHK2 sensors, IREα and PERK independent of the accumulation 52,96 have shown effects on myeloma cell proliferation and of ER luminal misfolded proteins . Thus, the basis of 5,23 viability . In response to ABC294640, a dual dihy- synergy likely arises from each drug activating ER stress droceramide desaturase and SPHK2 inhibitor, loss-of- via distinct mechanisms, culminating in terminal UPR myeloma cell viability was associated with degradation activation and apoptosis (Fig. 3). of c-Myc and Mcl-1, similar to the phenotype observed by Wallington-Beddoe et al. in ALL . The loss-of-Mcl-1, Conclusions the crucial Bcl-2 family member in myeloma ,in It is well known that sphingolipids are key mediators of response to ABC29460 appears as a consequence of Noxa cell fate and can rapidly fluctuate in response to drug Official journal of the Cell Death Differentiation Association Lewis et al. Cell Death Discovery (2019) 5:4 Page 9 of 11 Received: 2 June 2018 Accepted: 12 June 2018 treatment. The emerging findings from multiple haema- tological malignancies implicating rewiring of sphingoli- pid metabolism from different enzymes in the metabolic pathway, such as SPHKs, AC and GCS, highlights References multiple therapeutic angles to induce ceramide accumu- 1. Hochhaus, A. et al. Long-term outcomes of imatinib treatment for chronic lation. While the majority of drugs targeting these myeloid leukemia. N. Engl. J. Med. 376,917–927 (2017). 2. Biswal, S. S., Datta, K., Acquaah-Mensah, G. K. & Kehrer, J. P. 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Pathol. 235, intensive chemotherapy: 1-year outcomes. Blood 130, 890 (2017). 113–124 (2015). Official journal of the Cell Death Differentiation Association http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cell Death Discovery Springer Journals

Targeting sphingolipid metabolism as an approach for combination therapies in haematological malignancies

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Springer Journals
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Copyright © 2018 by The Author(s)
Subject
Life Sciences; Life Sciences, general; Biochemistry, general; Cell Biology; Stem Cells; Apoptosis; Cell Cycle Analysis
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2058-7716
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10.1038/s41420-018-0075-0
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Abstract

Conventional chemotherapy-based drug combinations have, until recently, been the backbone of most therapeutic strategies for cancer. In a time of emerging rationale drug development, targeted therapies are beginning to be added to traditional chemotherapeutics to synergistically enhance clinical responses. Of note, the importance of pro- apoptotic ceramide in mediating the anti-cancer effects of these therapies is becoming more apparent. Furthermore, reduced cellular ceramide in favour of pro-survival sphingolipids correlates with tumorigenesis and most importantly, drug resistance. Thus, agents that manipulate sphingolipid metabolism have been explored as potential anti-cancer agents and have recently demonstrated exciting potential to augment the efficacy of anti-cancer therapeutics. This review examines the biology underpinning these observations and the potential use of sphingolipid manipulating agents in the context of existing and emerging therapies for haematological malignancies. Facts � Efficacy of many chemotherapeutics and targeted therapies is dictated by cellular ceramide levels. � Oncogene activation skews sphingolipid metabolism to favour the production of pro-survival sphingolipids. � Inhibitors of enzymes involved in ceramide metabolism exhibit promise in the relapsed-refractory setting. � Anti-cancer activity of sphingosine kinase inhibitors provides several options for new drug combinations. Open Questions � What other clinically utilised drugs rely on increases in ceramide levels for their efficacy and can they be effectively partnered with other ceramide inducing agents? � How does ceramide modulate the Bcl-2 family proteins, Mcl-1 and Bcl-2? � Are sphingolipid enzyme inhibitors best suited in the frontline or relapsed-refractory setting? Introduction undergone little change over the last 30 years. The success Frontline chemotherapeutic regimens for the majority of tyrosine-kinase inhibitors (TKI) in chronic myeloid of haematological malignancies have, until recently, leukaemia (CML) has significantly increased the 10-year survival rate to 83%, enabling some patients to cease therapy (~40%) and achieve long-term remission Correspondence: Stuart M. Pitson (stuart.pitson@unisa.edu.au) (~40% >3 years) . However, unlike CML which is driven Centre for Cancer Biology, University of South Australia and SA Pathology, UniSA CRI Building, North Terrace, Adelaide, SA 5001, Australia solely by the BCR-ABL oncogene, most blood cancers are Adelaide Medical School, University of Adelaide, Adelaide, SA 5000, Australia more genetically heterogeneous displaying complex clonal Full list of author information is available at the end of the article. These authors contributed equally: Jason A. Powell, Stuart M. Pitson Edited by: A. Rufini © The Author(s) 2018. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Lewis et al. Cell Death Discovery (2019) 5:4 Page 2 of 11 Fig. 1 Overview of the sphingolipid cycle. The pleotropic nature of ceramide allows a promotion of multiple cellular fates including survival, migration and angiogenesis. Furthermore this also prevents a lethal accumulation of apoptotic sphingolipids (red) such as ceramide by maintaining a balance of pro-survival lipids (blue). Due to the propensity of transformed cells to deplete ceramide by increasing expression of enzymes, such as SPHK1/2 and GCS, inhibitors targeting these enzymes have exhibited therapeutic potential by tipping the balance to favour ceramide accumulation and promote cell death. architecture with conventional chemotherapeutic agents palmitoyl-CoA by the rate-determining enzyme, serine remaining the backbone of most therapy regimens. One palmitoyltransferase to 3-keto-dihydrosphingosine mechanism whereby chemotherapy induces apoptosis in (Fig. 1) . Following further modification to dihydro- malignant cells is through increases in the cellular levels sphingosine, ceramide synthases convert dihydro- 2,3 of the pro-apoptotic sphingolipid, ceramide . Sphingo- sphingosine to dihydroceramide which are then lipids are a class of lipids that can exert pleotropic cell desaturated to generate ceramides . From here, these signalling effects. Ceramide is a central component of ceramides can be modified to various sphingolipid species sphingolipid metabolism that is tightly regulated due to its that modulate membrane composition and signal trans- pro-apoptotic effects (Fig. 1). Enzymes involved in the duction. For example, ceramides can be glycosylated by conversion of ceramide to other sphingolipids have been glucosylceramide synthase to glucosylceramides which implicated in drug resistance by depleting ceramide to can serve as an intermediary for other glycosphingolipids, produce pro-survival sphingolipids (Table 1). Indeed, phosphorylated by ceramide kinase, modified by the inhibitors targeting these enzymes have been shown to addition of phosphocholine by sphingomyelinase (SMase) induce cell death through inducing the accumulation of to form sphingomyelins, or deacylated by ceramidases to 4–7 lethal levels of ceramide . Thus, manipulating sphin- form sphingosine and subsequently, through the action of golipid metabolism has shown promise in combination sphingosine kinases (SPHKs), generate sphingosine-1- with conventional chemotherapy, as well as with novel phosphate (S1P) (Fig. 1) . Notably, considerable evidence agents. In this review, we highlight the literature that implicates important roles for several of these sphingoli- examines how targeting sphingolipid metabolism shows pid metabolic enzymes in tumorigenesis and resistance to considerable promise for chemo-sensitising patients with therapy in haematological malignancies (Table 1). blood cancers. Sphingosine kinases The sphingolipid cycle The “sphingolipid rheostat” is a concept that describes While originally assumed to just play an integral role in cell fate as a balance between pro-apoptotic ceramide and 9,10 the structure of the cell membrane, sphingolipids have pro-survival S1P . As the SPHKs are critical in the only been found to be prominent players in cell signalling, exit point for degradation of sphingolipids, via conversion capable of exerting a myriad of cell responses. De of sphingosine to S1P, and then its degradation by S1P novo sphingolipid production commences at the endo- lyase, these enzymes represent one of the key players in plasmic reticulum with the condensation of serine and maintaining ceramide levels (Fig. 1). Furthermore, S1P Official journal of the Cell Death Differentiation Association Lewis et al. Cell Death Discovery (2019) 5:4 Page 3 of 11 Table 1 Role of sphingolipid enzymes in haematological Activated SPHK1 subsequently translocates to the plasma malignancies membrane, via an interaction with calcium and integrin binding protein 1 (CIB1), to convert sphingosine to Enzyme Malignancy Role 12–14 S1P . Due to the frequency with which hyperactiva- tion of the RAS pathway occurs in cancer, constitutive Acid ceramidase AML Increased expression in patient samples. phosphorylation and activation of SPHK1 in cancer is Modulates Mcl-1 expression in a post- likely to be common, promoting drug resistance by not translational manner only depleting ceramide levels but also promoting pro- Ceramide AML Suppressed by FLT3 signalling. Mediates 9,15,16 survival signalling . Furthermore, drug resistant cell synthase cytotoxicity of FLT3 inhibitors by lines have been shown to also exhibit increased SPHK1 induction of lethal mitophagy activity and attenuate increases in ceramide which would 15–17 Glucosylceramide AML Overexpressed in chemotherapy otherwise promote apoptosis . Several studies have 29,30 synthase resistance cell lines shown that targeting SPHK1 can resensitise cells to che- CLL Upregulated in response to B-cell motherapy, by blocking the conversion of sphingosine to 15, receptor stimulation S1P and generating a bolus of pro-apoptotic ceramide 16,18 98 . Lymphoma Potential role in tumour initiation Despite catalysing the same reaction, characterising Myeloma Potential role in tumour initiation SPHK2 has proved more elusive with conflicting literature 19,20 Sphingosine AML Overexpressed in patient samples. on its role in tumorigenesis . Nevertheless, targeting kinase 1 Increases drug resistance to SPHK2 has shown promise in a number of malignancies, chemotherapy and ceramide inducing including breast cancer , acute lymphoblastic leukaemia 16,18 22 5,23 strategies (ALL) and myeloma . ALL Overexpressed in patient samples Ceramidase CML Overexpressed in Imatinib-resistant cell Breakdown of ceramide to sphingosine is mediated by lines . Upregulates Mcl-1 in a BCR-ABL ceramidases, with several homologues described that dependent manner . Represses PP2A to function in acidic, neutral, or alkaline pH. The most promote BCR-ABL stability characterised form, acid ceramidase (AC) is thought to Sphingosine ALL Promotes B-ALL disease progression. localise to acidic compartments, such as lysosomes . The kinase 2 Inhibits histone deacetylases to promote degradation of ceramide by AC has highlighted a potential Myc expression mechanism for drug resistance by degrading the bolus of Myeloma Upregulated in cell lines and patient ceramide induced by cancer treatments. Overexpression 5,23 samples of AC has been reported in several solid tumours in addition to acute myeloid leukaemia (AML) . Pre-clinical AML acute myeloid leukaemia, B-ALL B cell acute lymphoblastic leukaemia, BCR- ABL Breakpoint cluster region–Abelson murine leukaemia viral oncogene studies utilising AC inhibitors have shown efficacy in homolog 1, CLL chronic lymphocytic leukaemia, FLT3 FMS-like tyrosine kinase 26,27 resensitising cells to chemotherapeutics . Although no 3, PP2A protein phosphatase 2A direct AC inhibitors are currently under investigation in clinical trials, interestingly, the clinically available oes- generation by SPHK also directly promotes cell survival as trogen receptor antagonist, Tamoxifen has demonstrated well as activating oncogenic signalling pathways by acting AC inhibition, warranting potential investigation as an as both an intracellular second messenger, and as a ligand adjuvant for chemotherapy regimens . for a family of five S1P-selective G-protein-coupled receptors . Glucosylceramide synthase Although very similar enzymes, some sequence and Glucosylceramide synthesis is the first step in the gen- presumed structural diversity between the two human eration of complex glycosphingolipids (Fig. 1). Much of SPHKs, SPHK1 and SPHK2, is thought to drive partially the interest in glucosylceramide has focussed on its role as different biological functions of these enzymes .Itis a “sink” for ceramide, with the action of glucosylceramide generally accepted that SPHK1 is associated with a pro- synthase (GCS) shunting ceramide through glucosylcer- tumorigenic role. Under homeostatic conditions, basal amide and the glycosphingolipid pathway. It is no surprise SPHK1 activity is thought to maintain ceramide levels that the removal of ceramide through this pathway in the absence of stimuli to prevent inappropriate has been proposed as a mechanism of drug resistance cell death . Activation of SPHK1 can occur via the and multiple studies have identified GCS as a therapeutic RAS pathway with extracellular signal-regulated kinases target by preventing the accumulation of lethal levels 12 29–32 1/2 (ERK1/2) phosphorylating SPHK1 at Serine 225 . of ceramide . The approval of the GCS inhibitor, Official journal of the Cell Death Differentiation Association Lewis et al. Cell Death Discovery (2019) 5:4 Page 4 of 11 Eliglustat, for clinical use in the lysosomal storage dis- inducing factor and second mitochondria-derived acti- order, Gaucher’s disease, suggests that GCS inhibition is vator of caspases (SMAC) . well tolerated in humans and could provide a means to investigate combinational studies of GCS inhibition with Targeting perturbations in sphingolipid chemotherapeutics and targeted agents. metabolism in haematological malignancies Clearly, sphingolipid metabolism is frequently dysre- Ceramide synthase gulated in haematological malignancies, and can confer In mammals, six different ceramide synthases (CerS1-6) resistance to many drugs currently employed to treat have been recognized, each capable of generating varying these diseases. Thus, there appears potential therapeutic ceramide species of differing fatty acyl chain lengths in benefit in combining sphingolipid modulators with clin- both the de novo sphingolipid pathway as well as the ical chemotherapeutics and novel therapies for the treat- conversion of sphingosine to ceramides in the so-called ment of a range of blood cancers. sphingolipid salvage pathway (Fig. 1) . Several studies have demonstrated that CerS activity is crucial to the Chronic myeloid leukaemia cancer cell killing efficacy of chemotherapeutics , The BCR-ABL inhibitor Imatinib and subsequent TKIs, tumour necrosis factor-related apoptosis-inducing ligand Dasatinib and Nilotinib have dramatically improved sur- 36 37 (TRAIL)-induced apoptosis , radiation and kinase vival rates for CML, with some patients even dis- 38,39 inhibitors . Although activation of CerS presents an continuing treatment due to prolonged molecular 55,56 attractive therapeutic target, CerS regulation remains remission status . The emergence of ATP binding site poorly understood . mutations within BCR-ABL, such as the T315I mutant, however, confers resistance to most of these ATP- Cellular ceramides are increased by chemotherapy competitive inhibitors, and presents a key therapeutic Chemotherapy frequently promotes the accumulation issue . The recent development of allosteric BCR-ABL of ceramide, which appears to contribute to induction of inhibitors, GNF-2 and ABL001 may provide an alternative 41–43 58 cancer cell death . While the mechanisms responsible option in the future to prevent this . Analysis of GNF-2 for this induction of ceramide depends on the che- treated cells revealed an increase in ceramide levels sug- motherapy employed, numerous studies have observed gesting BCR-ABL may suppress ceramide synthesis . chemotherapy-induced activation of CerS and acid Augmentation of ceramide levels using the glucosylcer- 41–44 SMase, as well as inhibition of AC amide synthase inhibitor D-threo-l-phenyl-2-decanoyla- . Notably, among the genes activated by p53 in response to chemotherapy, mino-3-morpholino-1-propanol (PDMP), enhanced 37 45 46 CerS5 , CerS6 and neutral SMase are prominent. apoptosis and resensitised T315I mutant CML cells to There is also evidence to suggest that the cysteine pro- both Imatinib and Nilotinib suggesting co-targeting tease, Cathepsin B can degrade SK1 following p53 upre- BCR-ABL with sphingolipid modulating agents may be a gulation by genotoxic stress, increasing ceramide levels . novel strategy to further enhance the efficacy of TKI Thus, with the widespread effect of chemotherapy treatment. and p53 on manipulating sphingolipid metabolism, direct Comparative analysis of Imatinib sensitive and resistant modulation of sphingolipid enzymes in combination K562 CML cells revealed enhanced S1P generation and with current agents may provide improved clinical reduced ceramide levels as a consequence of SPHK1 outcomes. upregulation compared to parental Imatinib sensitive K562 cells , suggesting a potential role for SPHK1 in Mechanisms of ceramide-induced cell death Imatinib resistance. Notably, overexpression of SPHK1 Ceramide exerts its tumour suppressive activities blocked the cytotoxic effects of Imatinib in sensitive K562 through multiple mechanisms, including activation of cells recapitulating the phenotype of Imatinib resistance . protein phosphatases 1 (PP1) and 2A (PP2A) , and Findings by Li et al. corroborated this relationship with 49 50 suppression of oncogenes such as Akt , c-Myc and BCR-ABL upregulating SPHK1 activity through MAPK, Bcr–Abl . Perturbation of sphingolipid homeostasis and PI3K and JAK2 signalling suggesting a positive feedback ceramide accumulation within cell membranes has loop . Genetic and chemical targeting of SPHK1 also also been reported to invoke pro-apoptotic signalling uncovered a positive relationship between SPHK1 and through activation of the unfolded protein response , anti-apoptotic protein Mcl-1, whereby overexpression of 52 39,53 autophagy and mitophagy . Furthermore, there is SPHK1 increased Mcl-1 levels in a BCR-ABL dependent strong evidence suggesting that ceramide can directly manner . initiate apoptosis by the formation of channels within the The precise mechanism between SPHK1 and BCR- mitochondrial outer membrane, capable of facilitating ABL stability was later uncovered with S1P receptor 2 the release of proteins, such as cytochrome c, apoptosis- (S1P ) supressing PP2A-mediated de-phosphorylation Official journal of the Cell Death Differentiation Association Lewis et al. Cell Death Discovery (2019) 5:4 Page 5 of 11 51 68 and subsequent degradation of BCR-ABL . Targeting kinase (BTK) activity . Notably, treatment of CLL cells S1P/S1P signalling resensitised Imatinib-resistant K562 with the PI3Kδ inhibitor, Idelalisib or BTK inhibitor, 51 69 cells and T315I patient blasts to BCR-ABL inhibition . Ibrutinib which are currently FDA approved for CLL , Notably, elevated SPHK1 and S1P mRNA levels were abrogated increases in GCS transcription and synergised found in T315I patient blasts used in this study which with the first generation Bcl-2/Bcl-xl inhibitor ABT- 7,67,68 raises the question as to whether there is a correlation 737 . These data suggest that combining ceramide- between these genes and BCR-ABL mutational status . inducing strategies with Bcl-2 inhibitors may exhibit BCR-ABL inhibitors, Dasatinib, Nilotinib and GFN-2 synergistic activity in CLL. have also been shown to induce transcription of various A recent study by Dielschneider et al. investigating the 59,61,62 CerS genes to upregulate ceramide production . use of “lysosomal penetrating” or lysosomotropic agents Intriguingly, Dasatinib treatment was associated with in CLL uncovered an increase in S1P phosphatase 1 increases in CerS2,5,6 whereas Nilotinib was associated (SPP1) and sphingosine in primary CLL patient samples 61,62 with increases in Cers5 expression . Combining either compared to normal B cells. Lysomotropic agents accu- Dasatinib or Nilotinib with the GCS inhibitor, PDMP, mulate within lysosomes, promoting the release of lyso- augmented apoptosis, presumably through synergistic somal contents such as cathepsins and the initiation 61 71 increases in ceramide levels . The interplay between of non-apoptotic cell death . As a natural detergent BCR-ABL, SPHK1 and CerS highlights the oncogenic and lysomotropic agent, sphingosine has been shown potential of BCR-ABL in skewing sphingolipid metabo- to induce lysosome destabilisation and thus may lism to favour a pro-survival phenotype, and highlights a explain the susceptibility of CLL cells to lysomotropic potential therapeutic benefit for combining ceramide agents . The addition of either sphingosine or SKI- inducers with BCR-ABL inhibitors. II augmented the apoptotic response of the lysosomo- tropic agent Siramesine, against primary CLL cells . The Chronic lymphocytic leukaemia clinical relevance of the above findings by Dielschneider Chronic lymphocytic leukaemia (CLL) is an indolent et al. are intriguing particularly with reports of the anti- form of leukaemia with some patients not requiring CD20 antibody Obinutuzumab inducing lysosomal per- treatment in their lifetime . CLL patients with unfa- meabilisation in B-cell malignancies, highlighting its vourable genetic factors such as 17p deletion (del(17p)) potential use in combination with ceramide/sphingosine- however respond poorly to frontline therapy in part due generating agents . to the absence of p53 . The addition of the Bcl-2 inhi- bitor, Venetoclax has been a paradigm shift for p53 null Acute lymphoblastic leukaemia CLL patients with 79% of relapsed/refractory patients in a There is disparity in treatment outcome for ALL with phase II clinical trial responding, 20% of whom exhibited cure rates for adults at 30–40% despite paediatric cases 65 74 undetectable disease by flow cytometry . As always, being closer to 90% . Poor risk subtypes such as Phila- resistance to drugs remains a problem with other Bcl-2 delphia (Ph)-like and Ph positive ALL are frequently family members such as Mcl-1, identified as a marker of observed in adults (~50%) and typically respond poorly to 66 75 resistance to Venetoclax . Although still effective in CLL chemotherapy . The addition of a TKI to standard che- patients, identifying other drugs to use alongside Vene- motherapy for Ph positive patients has improved treat- toclax and prevent the emergence of resistance is crucial. ment response and survival rates for many of these In the context of sphingolipid metabolism, the Bcl-2/ patients with initial studies showing complete haemato- Bcl-xl inhibitor Navitoclax was found to enhance CerS logical response in ~90% of patients . Like CML, activity and increase C16 ceramide synthesis. Although, relapsed Ph positive ALL patients exhibit similar muta- the mechanism remains to be confirmed, the authors to tions in BCR-ABL that impart TKI resistance, requiring a proposed that Bcl-2 inhibition allows Bak to interact with move to second and third generation BCR-ABL inhibitors. CerS5 or 6 resulting in the production of C16 ceramide . A potential role for the SPHKs in ALL was first high- Furthermore, combining Navitoclax with either the GCS lighted using studies with the pan SPHK inhibitor, SKI-II, inhibitor, PDMP, or the SPHK inhibitor, SKI-II-induced in combination with the commonly used chemother- 7 77 synergistic increases in ceramide and cell death . apeutic Vincristine . SKI-II treatment induced cell death Interestingly, B-cell receptor (BCR) signalling in pri- in ALL cell lines and primary lymphoblasts. As expected, mary CLL cells has been shown to induce glucosylcer- inhibition of the SPHKs and the accompanying increase in amide generation, potentially blunting the efficacy of ceramide synergised with Vincristine treatment . How- Rituximab treatment . Schwamb et al. proposed that ever, combining the SPHK2 inhibitor, ABC294640 with BCR signalling stimulates GCS transcription upon IgM Doxorubicin or Vincristine elicited only an additive effect treatment of primary CLL cells in a manner dependent on suggesting the synergy observed may be a consequence of phosphoinositide 3-kinase (PI3K)δ and Bruton’s tyrosine SPHK1 inhibition . Official journal of the Cell Death Differentiation Association Lewis et al. Cell Death Discovery (2019) 5:4 Page 6 of 11 A role for SPHK1 in contributing to the development of dependent cell death implicating mitochondrial mediated BCR-ABL driven ALL has been described. Gene expres- apoptosis . Although, LCL-204 appeared to display some sion data from two separate patient cohorts revealed a anti-leukaemic activity in patient-derived xenografts significant increase in SPHK1 expression in BCR-ABL in vivo, the assessment of circulating blasts within the positive ALL compared with BCR-ABL negative cases, periphery as opposed to bone marrow makes it difficult to highlighting a potential relationship that had previously draw conclusions from these studies. Overexpression of 17,60 been described in CML . Deletion of SPHK1 in BCR- AC correlated with an increase in Mcl-1 expression and ABL positive, but not BCR-ABL negative murine ALL blunted the cytotoxic effects of Bcl-2/Bcl-X inhibitor, models delayed disease incidence implicating SPHK1 as a ABT-737. Whilst Tan et al. demonstrate that AC inhibi- significant player in BCR-ABL driven ALL . Further- tion results in proteasome-mediated degradation of Mcl- more, combining SPHK inhibitors, SK1-I, SKI-II or 1, the exact mechanism of how AC upregulates ABC294640 with Imatinib also induced synergistic cell Mcl-1 was not further explored. Thus, the targeting of death in BCR-ABL positive cell lines which collectively Mcl-1 through AC inhibition warrants further investiga- warrants further investigation . tion in combination with Bcl-2 inhibitor, Venetoclax. Enhanced SPHK2 protein levels and activity in ALL Chemoresistant AML cells were also susceptible to cell patient samples and cell lines when compared with nor- death induced by AC inhibition which was correlated with 22 4 mal B-cell progenitors has also been reported . SPHK2 the increase in cellular ceramide levels . Although pre- inhibition was associated with reductions in histone liminary, these findings serve as a basis for further acetylation of the c-Myc promoter representing new evi- investigating the potential for AC inhibitors as chemo- dence for an oncogenic role for SPHK2 . Reductions resensitisers in the relapse/refractory setting. in c-Myc expression were observed in BCR-ABL The oestrogen receptor antagonist, Tamoxifen has also transformed B-ALL cells from SPHK2 knockout mice, demonstrated potent inhibition (IC ~1 μM) of GCS and translating into increased overall survival in vivo . These has demonstrated anti-leukaemic activity. Work by pre-clinical studies may provide the impetus to assess Morad et al. utilising short chain ceramides in combina- the addition of ABC294640 alongside BCR-ABL inhibitors tion with Tamoxifen revealed synergistic cell death in in patients with Ph positive ALL. AML cell lines as a consequence of mitochondrial meta- bolic collapse typified by decreases in ATP levels and Acute myeloid leukaemia glycolytic flux . Tamoxifen treatment prevented the Among drugs in the development pipeline for AML, accumulation of glucosylceramide species, in agreeance selective FLT3 inhibitors Crenolanib and ASP2215 have with the proposed role of these molecules as a “sink” for exhibited impressive single-agent activity (ORR 52%, CR excess cellular ceramide . Besides the demonstrated 41%) in a phase I/II study of relapsed/refractory AML . effects on GCS, Tamoxifen has also been reported to Recently, work by Dany et al. examined the mechanism of inhibit AC which may contribute to its cytotoxic effects . action of FLT3 inhibitors, including Crenolanib, and Nethertheless, the off-target inhibition of GCS by observed reductions in C18-ceramide levels in FLT3 Tamoxifen, presents an interesting therapeutic angle positive AML patient blasts . The authors attributed this given its approval for hormone-dependent breast cancer, to striking reduction in CerS1 mRNA levels, suggesting an potentially allowing fast track approval as adjuvant ther- inverse relationship between FLT3 activity and CerS1 apy in combination with chemotherapy. expression. Restoration of CerS1 upon treatment with Confirmation of SPHK1 as a therapeutic target in AML FLT3 inhibition resulted in mitophagy-dependent cell has been established by several groups using both genetic death suggesting repression of ceramide synthesis may be knockdown of SPHK1 and chemical SPHK1 inhibitors. an important step in AML pathogenesis. Intriguingly, a Chemotherapy resistant AML cell lines exhibited a lack of mitochondrial targeted ceramide analogue effectively ceramide generation upon drug treatment suggesting an suppressed FLT3 inhibitor resistant AML patient samples involvement of SPHK1 as a mediator of drug resistance . in vivo suggesting the reactivation of mitochondrial Overexpression of SPHK1 in chemo-sensitive AML cell ceramide synthesis downstream of FLT3 signalling may lines imparted resistance to chemotherapeutics confirming 16,18 be beneficial in overriding FLT3 resistance. SPHK1 as a marker of drug resistance in AML . Genetic Several groups have observed changes in AC in solid and chemical inhibition of SPHK1 in AML cell lines and 26,27,80 84 tumours , with its role in AML recently described. primary patient samples induced cell death and synergised Upregulation of AC mRNA and activity was observed in with chemotherapeutic agents . Interestingly, SPHK1 AML patient samples compared with normal CD34 inhibition-induced synergistic cell death with cytarabine in 4 18 bone marrow cells . Treatment of AML cell lines with the the refractory leukaemic initiating cell (LIC) population . AC inhibitor LCL-204 was associated with rapid loss of Although these synergistic effects require further in vivo the pro-survival Bcl-2 family protein, Mcl-1 and caspase evaluation, the findings suggest addition of an SPHK1 Official journal of the Cell Death Differentiation Association Lewis et al. Cell Death Discovery (2019) 5:4 Page 7 of 11 Fig. 2 Targeting sphingolipid metabolism in AML. An overview of targeting sphingolipid enzymes (red) in combination with pre-clinical or clinically utilised drugs (blue) in AML. inhibitor to a standard chemotherapeutic regimen has the expression, as well as the synergy observed with Bcl-2 7,18 potential to greatly enhance clinical responses and reduce inhibitors , provides considerable impetus to further relapse rates by targeting the LIC population. explore how ceramide modulates Mcl-1 stability. Recently, a link between SPHK1 and Mcl-1 was eluci- Targeting of S1P in AML has shown to induce loss-of- dated by Powell et al. whereby SPHK1 inhibition resulted in Mcl-1 and synergised with Bcl-2 inhibition recapitulating 18 18 Mcl-1 degradation . Loss-of-Mcl-1 coincided with induc- the observations with SPHK1 inhibition . A link between tion of BH3-only proteins, particularly Noxa, a known S1P and Mcl-1 stability has been largely overlooked in inducer of Mcl-1 degradation . As Mcl-1 is a marker of the context of cancer biology. We speculate that a change resistance to Bcl-2 inhibitor, Venetoclax, this link highlights in Mcl-1 phosphorylation may be responsible for loss-of- a new angle to target Mcl-1 and enhance the efficacy of Mcl-1 associated with S1P inhibition highlighting Venetoclax which is exhibiting impressive single-agent potential involvement of PP2A based on findings observed activity (ORR 79%) in CLL, a malignancy highly depen- in CML . Furthermore, this suggests that the loss-of- dent on Bcl-2 but only exhibited modest single-agent SPHK1 and its effect on Mcl-1 is two-pronged with activity in AML (ORR 19%) . Follow-up trials with Vene- reduced S1P/S1P signalling and the accumulation of toclax in combination with chemotherapy and hypo- ceramide due to the lack of sphingosine processing by methylating agents are currently ongoing with initial reports SPHK1. Despite the lack of targeted therapies in wide- 87,88 showing promising results . Pre-clinical evidence com- spread clinical use, the potentially broad applicability of bining SPHK1 and Bcl-2 inhibition showed synergistic cell inhibitors of sphingolipid metabolism in AML may pro- death in AML cell lines by targeting both Mcl-1 and Bcl-2 . vide significant benefit in a malignancy whose treatment Although, the exact mechanism is currently unknown, the options are reliant on ceramide accumulation for their 4 18 work from both Tan et al. andPowelletal. , suggests efficacy (Fig. 2). ceramide and S1P regulate Mcl-1 degradation. A link between ceramide and resistance to Bcl-2 tar- Multiple myeloma geting strategies has previously been identified in small The repertoire of novel therapies to treat multiple cell lung carcinoma which could explain the synergy myeloma has markedly increased over the past two dec- between the two drugs . Gene correlation analysis for ades. Of these novel therapies, the proteasome inhibitors Navitoclax resistance, identified an atypical Bcl-2 protein, (PIs), particularly Bortezomib (Velcade), have resulted in a Bcl-rambo, as a direct inhibitor of CerS2 and −6 . This marked improvement in overall response and median finding supports in vitro studies demonstrating that the survival rates (from 3 to 6–7 years) . Sustained mono- GCS inhibitor, PDMP synergises with Navitoclax sug- clonal immunoglobulin production by myeloma plasma gesting ceramide augments Bcl-2 targeting strategies . cells induces endoplasmic reticulum (ER) stress and The ability of sphingolipid modulating agents such as activates the unfolded protein response (UPR) which aims 4 18 those directed against AC or SPHK1 to reduce Mcl-1 to reduce global protein translation and correct the Official journal of the Cell Death Differentiation Association Lewis et al. Cell Death Discovery (2019) 5:4 Page 8 of 11 Fig. 3 Exploiting sphingolipid synthesis to enhance the efficacy of proteasome inhibitors. Due to the constitutive production of immunoglobulin by malignant plasma cells, their reliance on the unfolded protein response (UPR) to prevent an accumulation of misfolded proteins with the endoplasmic reticulum (ER) for survival renders them susceptible to inducers of ER stress. As the site of de novo sphingolipid synthesis, accumulation of saturated lipids such as ceramide within the ER, induces a lipid dependent UPR, promoting apoptosis. 91 23 misfolded protein-induced stress placed on the ER .To upregulation , just as it has been shown for Bortezo- effect this, proteins that cannot be correctly folded are mib , providing evidence for combination therapies. As transported to the 26S proteasome for degradation, expected, the loss-of-Mcl-1 observed with ABC294640 highlighting the relevance of PIs, which block proteasome treatment sensitised myeloma cells to Venetoclax . activity and commit the myeloma cell to apoptosis due to Indeed, combination of the SPHK2 inhibitor K145 and the accumulation of misfolded proteins in the ER lumen Bortezomib has been shown to result in synergistic anti- 92 5 and activation of a terminal UPR . myeloma effects in vitro and in vivo . These findings are Unlike other haematological malignancies, SPHK2 supported by studies showing that changes in lipid appears to be the dominant SPHK isoform in mye- saturation in the ER membrane, potentially including the 5,23 loma . Both pharmacological inhibition of SPHK2 using accumulation of sphingolipids, can activate the UPR ABC294640 or K145, and genetic interference of SPHK2 sensors, IREα and PERK independent of the accumulation 52,96 have shown effects on myeloma cell proliferation and of ER luminal misfolded proteins . Thus, the basis of 5,23 viability . In response to ABC294640, a dual dihy- synergy likely arises from each drug activating ER stress droceramide desaturase and SPHK2 inhibitor, loss-of- via distinct mechanisms, culminating in terminal UPR myeloma cell viability was associated with degradation activation and apoptosis (Fig. 3). of c-Myc and Mcl-1, similar to the phenotype observed by Wallington-Beddoe et al. in ALL . The loss-of-Mcl-1, Conclusions the crucial Bcl-2 family member in myeloma ,in It is well known that sphingolipids are key mediators of response to ABC29460 appears as a consequence of Noxa cell fate and can rapidly fluctuate in response to drug Official journal of the Cell Death Differentiation Association Lewis et al. Cell Death Discovery (2019) 5:4 Page 9 of 11 Received: 2 June 2018 Accepted: 12 June 2018 treatment. The emerging findings from multiple haema- tological malignancies implicating rewiring of sphingoli- pid metabolism from different enzymes in the metabolic pathway, such as SPHKs, AC and GCS, highlights References multiple therapeutic angles to induce ceramide accumu- 1. Hochhaus, A. et al. Long-term outcomes of imatinib treatment for chronic lation. While the majority of drugs targeting these myeloid leukemia. N. Engl. J. Med. 376,917–927 (2017). 2. Biswal, S. S., Datta, K., Acquaah-Mensah, G. K. & Kehrer, J. P. 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