Abstract
A subset of Ras proteins, including N-Ras, depend on a palmitoylation/depalmitoylation cycle to regulate their subcellular trafficking and oncogenicity. General lipase inhibitors such as Palmostatin M block N-Ras depalmitoylation, but lack specificity and target several enzymes displaying depalmitoylase activity. Here, we describe ABD957, a potent and selective covalent inhibitor of the ABHD17 family of depalmitoylases, and show that this compound impairs N-Ras depalmitoylation in human acute myeloid leukemia (AML) cells. ABD957 produced partial effects on N-Ras palmitoylation compared to Palmostatin M, but was much more selective across the proteome, reflecting a plasma membrane-delineated action on dynamically palmitoylated proteins. Finally, ABD957 impaired N-Ras signaling and the growth of NRAS-mutant AML cells in a manner that synergizes with MEK inhibition. Our findings uncover a surprisingly restricted role for ABHD17 enzymes in modulating the N-Ras palmitoylation cycle and suggest that ABHD17 inhibitors may have value as targeted therapies for NRAS-mutant cancers.
RAS genes are the most common targets of dominant mutations in human cancer1, 2. There are three different RAS genes, which encode four highly homologous proteins (H-Ras, N-Ras, K-Ras4a, and K-Ras4b), and they are preferentially mutated in distinct tumor types1, 2. An elegant approach for directly targeting Ras oncoproteins involves designing covalent inhibitors targeting cysteine 12 of K-RasG12C 3-5. However, it is more challenging to apply this approach to oncogenic amino acid substitutions that introduce side chains lacking reactive nucleophiles. Given the difficulties inherent in directly targeting oncogenic Ras, inhibitors of Ras effector molecules such as PI3K, Akt, mTOR, and MEK are being pursued as an alternative therapeutic strategy6, 7.
Ras proteins share very high homology throughout most of their protein sequence with the exception of the C-terminal “hypervariable region” (HVR), which contains signals that specify post-translational modifications required for proper subcellular localization8. The HVR of all four isoforms terminates with a CAAX motif, where the cysteine is prenylated by the farnesyltransferase FTase (or FNTA/FNTB). This lipid modification provides weak membrane binding affinity that is stabilized by a second signal motif. For K-Ras4b, this is provided by a polybasic lysine domain9, 10. By contrast, H-Ras, N-Ras, and K-Ras4a are S-palmitoylated at cysteine(s) adjacent to the CAAX motif 8, 9, and a dynamic cycle of palmitoylation/depalmitoylation mediated by palmitoyl acyl transferase (PAT) and serine hydrolase (SH) enzymes has been shown to regulate H-and N-Ras trafficking, subcellular localization, and function11-13.
Of the 23 mammalian PATs, only DHHC9 has been directly shown to catalyze N-Ras palmitoylation in conjunction with GOLGA714, 15. The enzymes involved in depalmitoylating H- and N-Ras are presumed to be serine hydrolases based on studies showing that N-Ras depalmitoylation is blocked by the beta-lactone small molecules palmostatin B and M (Palm B and M)12, 16 or hexadecylfluorophosphonate (HDFP)17, as well as earlier work demonstrating that the yeast serine hydrolase APT1 (or, in humans, LYPLA1) can depalmitoylate H-Ras18. While LYPLA1 and LYPLA2 are inhibited by Palm B and M19, selective inhibitors or genetic knockdown of these serine hydrolases do not block the palmitoylation dynamics of N-Ras in cancer cells20, pointing to other enzymes being involved in this process. Using activity-based protein profiling (ABPP) methods, Lin and Conibear recently identified ABHD17 proteins, a poorly characterized group of three sequence-related serine hydrolases (ABHD17A, B, and C), as additional targets of Palm M and HDFP and showed that these enzymes can depalmitoylate N-Ras20. The ABHD17 enzymes are themselves palmitoylated on their N-termini, which promotes localization to the plasma membrane of cells21. Importantly, overexpression of ABHD17A was found to enhance N-Ras depalmitoylation and altered N-Ras subcellular localization in COS-7 cells, and, conversely, triple knockdown of ABHD17A/B/C by RNA interference impaired N-Ras palmitoylation turnover, albeit less effectively than Palm B, in HEK293T cells20.
Previous findings, taken together, support a potential role for ABHD17 enzymes in regulating N-Ras palmitoylation. However, ABHD17A, B, and C are all broadly expressed in most cell types, which makes characterization of their collective contribution to N-Ras palmitoylation challenging using genetic methods. It remains unclear, for instance, whether the partial blockade of N-Ras depalmitoylation observed in triple ABHD17A/B/C knockdown cells reflects an incomplete reduction in the expression of these enzymes or a contribution of other Palm B and M targets to the N-Ras palmitoylation cycle. Additionally, the studies performed to date relating ABHD17 enzymes to N-Ras depalmitoylation have occurred in heterologous cell types that do not depend on N-Ras as an oncogenic driver. Accordingly, it remains unknown whether ABHD17 enzymes regulate N-Ras palmitoylation in relevant biological systems and if disrupting these enzymes will affect the growth of NRAS mutant cancer cells. Finally, the broader potential substrate scope of ABHD17 enzymes beyond N-Ras and a handful of other palmitoylated proteins (e.g. PSD-9520, 22) remains unknown.
To address the aforementioned knowledge gaps, we describe herein a potent, selective, and cell-active covalent inhibitor of the ABHD17 enzymes and the characterization of its pharmacological effects in NRAS mutant cancer cell lines. We find that this compound – termed ABD957 – inhibits all three ABHD17 enzymes and partially impairs N-Ras depalmitoylation, ERK phosphorylation, and the growth of NRAS mutant cancer cells. ABD957 stabilizes N-Ras palmitoylation less completely than Palm M, but is also much more selective across the proteome, being restricted in its effects to plasma membrane-associated, dynamically palmitoylated proteins. ABD957 and Palm M also differentially affect the subcellular distribution of N-Ras, leading to the accumulation of this protein on the plasma membrane and intracellular membranes, respectively. Finally, we show that ABD957 synergizes with MEK inhibition to block NRAS mutant cancer cell growth. These data, taken together, indicate that ABHD17 enzymes perform a specialized function as plasma membrane-delineated S-depalmitoylases and that the chemical inhibition of these enzymes attenuates dynamic N-Ras depalmitoylation, resulting in impaired signaling and growth of NRAS mutant cancer cells.
Results
Discovery and characterization of ABHD17 inhibitors
The palmostatins (Palm B and Palm M)12, 16 and HDFP17 (Fig. 1a) are useful pharmacological tools for studying protein palmitoylation through blockade of depalmitoylase enzymes and, to date, remain the only reported inhibitors of ABHD17A/B/C20. HDFP is a broad-spectrum serine hydrolase inhibitor, targeting multiple depalmitoylases including LYPLA1/2, PPT1, ABHD10 and ABHD17A/B/C17. This compound13, 17, 23, along with other long-chain FPs24, 25, have been shown to preserve the palmitoylation state of several dynamically palmitoylated proteins, including Ras proteins17, 20, in mammalian cells. Palm B and M also inhibit several other serine hydrolases19, 20 and this lack of selectivity, along with presence of a metabolically labile beta-lactone, has limited the broad utility of these compounds in biological systems. Such factors motivated us to develop a more advanced chemical probe that selectively inhibits ABHD17 enzymes.
HDFP and palmostatins feature reactive electrophilic centers that inhibit serine hydrolases through covalent modification of the catalytic serine residue. While this feature likely contributes to a lack of specificity across the serine hydrolase class, more tempered electrophilic chemotypes, such as carbamates and ureas, have been shown to covalently inhibit individual serine hydrolases with excellent potency, selectivity, and cellular and in vivo activity26-32. The evaluation of candidate inhibitors of serine hydrolases has also benefited from the chemical proteomic technology activity-based protein profiling (ABPP), wherein activity-based probes showing broad reactivity with serine hydrolases are used to measure target engagement and selectivity of inhibitors in native biological systems33-35. We accordingly screened a serine hydrolase-directed compound library internally developed at Lundbeck La Jolla Research Center by gel-based ABPP in native mouse brain proteomes, where ABHD17 and other serine hydrolases can be visualized using a fluorescently (rhodamine) tagged fluorophosphonate (FP) activity-based probe. This screen furnished a piperazine-based pyrazole urea hit (1, Fig. 1b) that displayed moderate potency (IC50 [95% CI] = 0.93 [0.79-1.1] µM, Table 1 and Supplementary Fig. 1a, b) against human ABHD17B, assayed by gel-based ABPP in stably tranduced HEK293T cells.
Compound 1 represented an attractive starting point as an ABHD17 inhibitor due to its simple, chemically tractable core and the presence of a moderately electrophilic pyrazole urea group resembling those found in other advanced chemical probes that covalently inhibit serine hydrolases26, 30, 31, 36. We next used a biotinylated FP probe37 and quantitative, mass spectrometry (MS)-based ABPP31 to assess the selectivity of 1 in the proteome of the human NRAS mutant acute myeloid leukemia (AML) cell line OCI-AML338. Experiments performed with 1 and 10 µM of 1 confirmed simultaneous inhibition of all three A, B and C isoforms of ABHD17, as well as several additional serine hydrolases, including LYPLA1 and LYPLA2 (Fig. 1c and Supplementary Fig. 1c). We focused our initial optimization efforts on improving selectivity for ABHD17 enzymes, using cross-reactivity with human LYPLA1 as a guide, since this enzyme could be readily visualized by gel-based ABPP in ABHD17B-transduced HEK293T proteome (Supplementary Fig. 1b). We found that three modifications to 1 were sufficient to eliminate LYPLA1/2 activity while simultaneously improving inhibitory potency and selectivity for ABHD17s. First, introduction of a morpholino group to the benzyl moiety (compound 2, Fig. 1b) resulted in a ∼10-fold loss of potency for LYPLA1 activity without impairing activity against ABHD17B (Table 1). Secondly, shifting the sulfonamide from the 4- to the 3-position on the pyrazole leaving group (compound 3; Fig. 1b) diminished LYPLA1 activity further (IC50 ∼10 µM) and concurrently enhanced hABHD17B potency by ∼4-fold (IC50 [95% CI] = 0.25 [0.19-0.32] µM) (Table 1). MS-based ABPP experiments revealed that these modifications also had a favorable impact on global selectivity across the serine hydrolase family, removing off-targets such as ABHD11 (Fig. 1c and Supplementary Fig. 1c). Reasoning that increasing the steric demand around the electrophile could promote selectivity, as has been seen for other serine hydrolases28, 30, we introduced a methyl substituent on the piperazine staying group adjacent to the urea (compound 4, Fig. 1b), which further boosted potency (IC50 [95% CI] = 0.13 [0.11-0.16] µM, Table 1) and shifted the selectivity profile - eliminating PAFAH2, but introducing ABHD10, a recently identified mitochondrial depalmitoylase25, as an off-target (Fig. 1c and Supplementary Fig. 1c). Furthermore, 4 suffered from poor metabolic stability in human liver microsomes (482 µL/min/mg, Table 1) and, when tested in cells, did not produce sustained inhibition of ABHD17s over 72 h (Fig. 1d), a common endpoint used for cell-based assays that measure, for instance, proliferation.
Additional modifications to 4, specifically the introduction of a carboxymethyl-substituted diazobicyclo[3.2.1]octane in place of the morpholino group, furnished the more advanced chemical probe ABD957 (compound 5, Fig. 1b) that displayed much lower microsomal clearance (4.8 µL/min/mg, Table 1) while maintaining good potency for ABHD17B (IC50 [95% CI] = 0.20 [0.15-0.26] µM, Fig. 1e, Supplementary Fig. 1b, and Table 1). MS-based ABPP revealed that ABD957 exhibits excellent selectivity across the serine hydrolase class, including avoidance of ABHD10 as an off-target (Fig. 1c and Supplementary Fig. 1c). ABD957 did cross-react with a handful of other off-targets across the >60+ serine hydrolases assayed by MS-based ABPP, but, to our knowledge, none of these enzymes have been reported to display protein depalmitoylation activity. Recognizing that cellular potency and selectivity were more pertinent parameters for assessing the suitability of ABD957 as a chemical probe, we performed MS-based ABPP experiments on OCI-AML3 “ON” (described below) cells treated in situ with ABD957 (500 nM) for 2 h (Fig. 1f). ABD957 blocked the activities of ABHD17A, B, and C under these conditions with good overall selectivity, including no cross-reactivity with LYPLA1, LYPLA2, or ABHD10. While ABD957 cross-reacted with a handful of other serine hydrolases, including CES1/2, ABHD6 and ABHD13, the global selectivity was markedly improved compared to previous ABHD17 inhibitors, such as Palm M and HDFP, which, consistent with previous studies17, 20, caused widespread serine hydrolase inhibition in cells (Supplementary Fig. 1d). Importantly, ABD957 (1 µM) produced sustained cellular inhibition of ABHD17A/B/C for 72 h (Fig. 1d), indicating that this compound was well-suited for a diverse array of cell-based experiments.
ABHD17 inhibition attenuates N-Ras depalmitoylation in AML cells
To assess the impact of ABHD17 inhibition on N-Ras palmitoylation, we adapted a pulse-chase assay for measuring dynamic protein palmitoylation previously described by our lab17. We first generated sublines of OCI-AML3 cells in which we used RNA interference to reduce the expression of endogenous human N-Ras and then introduced into these cells either i) a murine GFP-N-RasG12D protein, or ii) a murine GFP-N-RasG12D variant where the palmitoylated HVR sequence was replaced with the non-palmitoylated HVR sequence of K-Ras4b (Supplementary Fig. 2). The two engineered cell lines expressing GFP-N-RasG12D and GFP-N-RasG12D, KRAS HVR proteins were termed “ON” and “ONK”, respectively. We next evaluated the effect of ABD957 and other compounds on N-Ras palmitoylation dynamics by pre-incubating ON or ONK cells with compounds or DMSO control for 1 h, then treating cells with the clickable palmitate analog 17-octadecynoic acid (17-ODYA21, 39, 40; 20 µM) for 1 h, followed by a 1 h chase period where the media was changed to remove 17-ODYA and supplemented with fresh inhibitor (Fig. 2a, left and middle panels). The palmitoylation state of N-Ras was then determined by anti-GFP immunoprecipitation, on-bead copper-catalyzed azide-alkyne cycloaddition (CuAAC)41, 42 to a rhodamine-azide (Rh-N3) reporter tag, and visualization of N-Ras palmitoylation signals by SDS-PAGE and in-gel fluorescence scanning (Fig. 2a, right lower panel). In this assay format, a compound that inhibited N-Ras depalmitoylation could be identified by comparing 17-ODYA labeling signals post-chase to a DMSO control sample. We also used a similar experimental protocol to survey the proteome-wide effects of candidate depalmitoylation inhibitors by performing CuAAC with Rh-N3 on lysates from 17-ODYA-treated cells (Fig. 2a, right upper panel).
Consistent with previous studies12, 16, 17, 20, we found that the promiscuous lipase inhibitors Palm M and HDFP near-completely preserved N-Ras palmitoylation following the chase period (Fig. 2b, c). ABD957 also protected N-Ras from depalmitoylation, but to a lesser degree than Palm M or HDFP (Fig. 2b, c). In contrast, the dual LYPLA1 and LYPLA2 inhibitor JJH25443 did not affect N-Ras palmitoylation dynamics, either when administered on its own or in combination with ABD957 (Fig 2b, c). As expected, palmitoylation signals were not observed in ONK cells (Fig. 2b, d), indicating that the GFP-N-RasG12D, KRAS HVR protein does not undergo palmitoylation in the pulse-chase experiment.
The partial stabilization of N-Ras palmitoylation observed with ABD957 was concentration-dependent and showed an estimated IC50 value of 29 nM (Fig. 2d, e), which is consistent with the inhibition of ABHD17A/B/C in cells treated with 500 nM of this compound, as measured by MS-ABPP (Fig. 1f). Proteome-wide surveys of palmitoylation dynamics by SDS-PAGE revealed further that, while both Palm M and HDFP broadly affected the palmitoylation state of proteins in ON cells, ABD957 had a much more limited impact on dynamically palmitoylated proteins (Supplementary Fig. 3a). Intrigued by the distinct profiles of different depalmitoylation inhibitors, we next evaluated the effect of these compounds on global protein palmitoylation using quantitative MS-based proteomics.
ABHD17 inhibition regulates the palmitoylation of plasma membrane proteins
We evaluated the impact of compounds on global palmitoylation dynamics using a similar pulse-chase protocol to that previously described17 and shown in Fig. 2a, where 17-ODYA-labeled proteins were conjugated by CuAAC to biotin-N3 to enable streptavidin enrichment of these labeled proteins followed by quantitative, multiplexed MS-based proteomics using tandem mass tags (TMT)44. We also required that the enrichment of 17-ODYA-labeled proteins show sensitivity to hydroxylamine treatment for designation as palmitoylated proteins, as reactivity with hydroxylamine is a characteristic feature of chemically labile thioester adducts formed between fatty acids and cysteine residues on proteins45. In total, 224 palmitoylated proteins were identified in ON cells (Supplementary Table 1). Consistent with past studies17, 23 only a modest subset (nineteen) of these proteins, which included N-Ras, displayed clear evidence of dynamic palmitoylation in pulse-chase experiments of ON cells, as reflected by at least two-fold reductions in 17-ODYA modification following the 1 h chase period (t0/t1 ≥ 2) (Fig. 3a, b, green and red proteins rightward of vertical dashed red line; and Supplementary Table 1). The palmitoylation states of most of these proteins were stabilized by treatment with Palm M (10 µM) (Fig. 3a, red proteins; and Supplementary Table 1). In contrast, ABD957 (500 nM) preserved the palmitoylation state of only a select few proteins, including N-Ras (Fig. 3b, red proteins). Another noteworthy difference between Palm M and ABD957-treated cells is that Palm M increased the apparent palmitoylation state of an additional set of proteins that otherwise did not show evidence of dynamic palmitoylation (Fig. 3a, blue proteins and Supplementary Fig. 4a). We are unsure of the mechanistic basis for these changes, but they point to a much broader effect of Palm M on the palmitoylated proteome compared to ABD957.
In agreement with our focused palmitoylation assays on N-Ras (Fig. 2b), the palmitoylation state of this protein was more completely preserved by Palm M compared to ABD957 (Fig. 3c). Notably, the quantification of N-Ras signals could be performed in ON cells using both peptides that are unique to N-Ras (relative to other Ras isoforms) and peptides from the fused GFP protein as a surrogate, and both sets of peptides provided similar data supporting the preservation of N-Ras palmitoylation by Palm M and ABD957 (Supplementary Fig. 4b and Supplementary Table 2). In addition to N-Ras, ABD957 substantially preserved the palmitoylation state of SCRIB (Fig. 3d), a tumor suppressor protein that depends on palmitoylation for proper plasma membrane localization and function46, and MPP6, a palmitoylated guanylate kinase that has been shown to localize to the basolateral plasma membrane of intestinal epithelial cells47. In the case of SCRIB, ABD957 produced a degree of protection of palmitoylation that matched that observed for Palm M (Fig. 3d).
When considering possible reasons for the more restricted effect of ABD957 on the palmitoylated proteome compared to Palm M, we noted that the three main proteins affected by ABD957 all reside at the plasma membrane (Fig. 3e). In contrast, dynamically palmitoylated proteins that were affected by Palm M, but not ABD957, localized to other subcellular compartments (e.g., Golgi (LYPLA1, TMEM59)48, 49, nucleus (NPM1, NUP210)50, 51, lysosome (LAPTM5)52, mitochondria (FAM49B, PTRH2)53, 54) (Fig. 3e), where they may not interface with plasma membrane-localized ABHD17 enzymes. One possible exception is the metalloproteinase ADAM10, which localizes to both the Golgi apparatus and plasma membrane55, and, for which the palmitoylation state was preserved by Palm M, but not ABD957. Our quantitative proteomic data thus indicate that the ABHD17 enzymes regulate the dynamic palmitoylation state of N-Ras and a discrete set of plasma membrane-localized proteins in leukemia cells.
ABD957 and Palm M differentially affect N-Ras localization in leukemia cells
Previous studies have shown that Palm B and M alter the subcellular localization of N-Ras, leading to redistribution of this protein from the plasma membrane to endomembranes12, 16, 56. This effect was also shown to depend on the presence of the N-Ras HVR in transduced primary hematopoietic cells56. Using live-cell confocal microscopy, we monitored GFP-N-Ras localization in ON cells following treatment with Palm M or ABD957. Consistent with previous studies12, 16, we found that Palm M promoted the time-dependent accumulation of N-Ras at endomembranes (Fig. 4a, upper panels). The intracellular N-Ras showed partially overlapping distribution with the Golgi marker N-acetylgalactosaminyltransferase (GALNT2, visualized as an RFP-tagged protein) (Fig. 4b and Supplementary Fig. 5). ABD957, on the other hand, promoted the retention of GFP-N-Ras at the plasma membrane without evidence of endomembrane redistribution (Fig 4a-c and Supplementary Fig. 5). The effect of ABD957 was selective for palmitoylated N-Ras, as it was observed in ON, but not ONK cells (Fig. 4c). These cell imaging results indicate that ABHD17 inhibition, in addition to partially protecting the palmitoylation state of N-Ras, leads to the accumulation of this protein at the plasma membrane, where ABHD17 enzymes are themselves localized21.
ABHD17 inhibition selectively blocks NRAS dependent cell growth and synergizes with the MEK inhibitor PD901
We next investigated how ABD957 affected N-Ras function and the growth of N-Ras-dependent cancer cells. We first addressed this question by measuring downstream signaling, specifically ERK phosphorylation, which we found was substantially blocked by both Palm M and ABD957 in NRAS mutant (and N-Ras-dependent) OCI-AML3 cells, but not in KRAS mutant (and N-Ras-independent) NB4 cells57 (Fig. 5a, b). In contrast, the MEK inhibitor, PD901, which functions downstream of Ras signaling, blocked ERK phosphorylation in both cell lines (Fig. 5a, b). The effect of Palm M on ERK phosphorylation was greater than ABD957, mirroring their respective impacts on N-Ras palmitoylation (Fig. 2 and 3).
The enhanced cellular stability of ABD957 (Fig. 1d) enabled further studies of its impact on cell proliferation, where we found that the compound reduced the growth of NRAS mutant AML cell lines (OCI-AML3, THP1, and HL60), but not KRAS mutant cell lines (NB4 and NOMO1) (Fig. 5c). The growth inhibitory effects of ABD957 were partial and plateaued at ∼500 nM (Fig. 5c) consistent with the near-complete inhibition of ABHD17 enzymes in AML cells treated with this concentration of the compound (see Fig. 1f). We next explored the impact of combining ABHD17 and MEK inhibitors by exposing ON and ONK cells to a range of ABD957 and PD901 concentrations. Using the Bliss Independence Model58 to assess potential drug interactions, we found that the combination of ABD957 and PD901 was substantially synergistic in ON cells, but not ONK cells (Fig. 5d, e), pointing to ABHD17 inhibition as a possible way to augment the therapeutic activity of MEK inhibitors in N-Ras-dependent cancers.
Discussion
The fundamental role that C-terminal lipid modifications play in regulating Ras signaling has stimulated several historical efforts to target the post-translational processing of Ras onco-proteins6, 59. In the 1990s, potent and selective farnesyltransferase (FTase) inhibitors were developed; however, these compounds did not effectively block Ras modification or signaling due to alternative processing of N-Ras and K-Ras by geranylgeranyl transferases60-64. More recently, the importance of an additional lipid modification – dynamic cysteine S-palmitoylation – for certain Ras oncoproteins, including N-Ras, has pointed to an alternative set of pharmacological targets for controlling Ras signaling in cancer cells7, 65. Biochemical and pharmacological studies with first-generation inhibitors of N-Ras depalmitoylation have implicated several enzymes in this process12, with more recent studies revealing a possible key contribution from the ABHD17 sub-group of serine hydrolases20.
The high sequence identity shared by ABHD17A, ABHD17B, and ABHD17C (>70%) and their co-expression in most cell types presents technical challenges for studying the net contribution of these enzymes to N-Ras palmitoylation, which, to date, has not been assessed in NRAS-mutant cancer cells. We have addressed this problem by creating ABD957, a pan-ABHD17 inhibitor that displays good potency, proteomic selectivity, and metabolic stability. Using ABD957, we show that the ABHD17 enzymes make a substantial, but partial contribution to N-Ras depalmitoylation in NRAS-mutant AML cells, which is in agreement with previous work in heterologous cell systems showing that RNAi interference-mediated knockdown of ABHD17A/B/C failed to completely arrest N-Ras dynamic palmitoylation20. Whether other targets of the more promiscuous inhibitors Palm M and HDFP account for the putative ABHD17-independent N-Ras depalmitoylation activity remains unknown, but our pharmacological studies testing ABD957 in combination with JJH254 would argue against additional contributions from LYPLA1 and LYPLA2. We also note that our broader proteomic investigations of Palm M identified several proteins with heightened palmitoylation, including those that were not dynamically palmitoylated over the time course of our pulse-chase experiments. This result suggests that Palm M may affect other cellular metabolic processes beyond protein depalmitoylation that in turn impact the net palmitoylation state of proteins in cells.
One of our most striking findings was the selectivity that ABD957 displayed for N-Ras across the broader palmitoylated proteome. Unlike Palm M, which affected the palmitoylation state of many proteins, ABD957 only preserved the palmitoylation state of three plasma membrane-associated proteins – N-Ras, SCRIB, and MPP6. Considering that most of the other dynamically palmitoylated proteins affected by Palm M, but not ABD957, localize to intracellular membrane compartments (Fig. 3e), our data support a model where ABHD17 enzymes regulate palmitoylated proteins exclusively at the plasma membrane. We do not know yet whether individual ABHD17 enzymes (A, B, or C) will show differential substrate specificities in cells, but, regardless, it is likely that the substrate scope of this family of depalmitoylases will expand as more cell types are treated with ABD957 and examined for their palmitoylation profiles. Indeed, the palmitoylation of the plasma membrane-associated, post-synaptic density protein PSD-95 (DLG4), which is predominantly expressed in neurons and was not detected in the AML cells examined herein, has also been shown to be regulated by ABHD17 enzymes20, 22. Conversely, SCRIB has been found, in other cell types, to be regulated by the depalmitoylase LYPLA266, indicating the potential for cellular context to also restrict the substrate scope of ABHD17 enzymes. With regards to other enzymes that may be responsible for regulating the palmitoylation state of intracellular proteins, we note that the serine hydrolase ABHD10 was recently identified as a depalmitoylase localized to the mitochondria25. Thus, emerging data point to the existence of multiple S-depalmitoylases that individually regulate dynamic protein palmitoylation in specific organelles of mammalian cells.
The general strategy of inhibiting the palmitoylation/depalmitoylation cycle of N-Ras has the advantage of selectively targeting cancer cells that depend on this oncoprotein for growth while preserving the functions of K-Ras4b in normal tissues. Our data indicate that ABHD17 inhibitors offer one potential way to achieve this objective. However, several important mechanistic questions about the interactions between ABHD17 enzymes and N-Ras palmitoylation and signaling remain to be answered. How, for instance, does ABD957-induced accumulation of palmitoylated N-Ras at the plasma membrane lead to perturbation in N-Ras signaling? Might, for instance, persistently palmitoylated N-Ras mislocalize, over time, to plasma membrane microdomains that are less productive for signal transduction? We also wonder whether the partial blockade of N-Ras signaling and cancer growth caused by ABHD17 inhibitors reflect maximal possible effects of the plasma membrane-delineated stabilization of N-Ras palmitoylation. Considering that Palm M more greatly affected N-Ras signaling (as measured by ERK phosphorylation), it is possible that redirecting N-Ras to intracellular membranes is required to more completely block signal transduction, an outcome that, based on our data, may not be achievable with ABHD17 blockade alone. On the other hand, the remarkably restricted impact of ABHD17 inhibitors on the palmitoylated proteome suggests that these compounds may perturb N-Ras signaling without gross effects on normal cell physiology. Such a profile may enable ABHD17 inhibitors to safely augment the activity of other anti-cancer agents, as we have shown for the MEK inhibitor PD901, to provide a compelling path for the targeted therapy of NRAS mutant cancers.
Contributions
J.R.R., R.M.S., N.A.Z., M.J.N., K.S., and B.F.C. conceived the project and wrote the paper. J.R.R. performed palmitoyl-proteomics experiments. R.M.S. perfomed NRAS palmitoylation assays. N.A.Z., J.R.R., A.J.F., A.I., A.L., M.P., and B.H. performed proliferation and NRAS signaling assays. T.W.H. performed imaging experiments. A.J.F. developed syngenic AML cell lines. N.N., C.L.H., and R.M.S. performed gel- and MS-ABPP experiments. N.N. and C.L.H. performed compound characterization. K.M.L. performed and analyzed MS-ABPP experiments and analyzed spectra. S.K.R. synthesized Palmostatin M. A.R.H. provided synthetic expertise and contributed to related studies not detailed in this paper. J.R.R., R.M.S., N.A.Z., T.W.H., K.M.L., and M.J.N. performed data analysis and visualization.
Supplementary Inforrmation
Included are Supplementary Methods, Supplementary Figures 1-6, including full-length versions of cropped gels and blots shown in Figures 1-5 and Supplementary Figures 1-5, Supplementary Tables 1 and 2.
Supplementary Figure 6. Full-length gels and blots corresponding to the indicated cropped gels and blots in other figures.
Acknowledgements
This work was supported by grants from the NIH (CA193994, CA231991, and CA72614), an American Cancer Society postdoctoral fellowship PF-18-217-01-CDD (J.R.R.), the Leukemia and Lymphoma Society (LLS Fellowship 5465-18 to N.A.Z.), and the Damon Runyon Cancer Research Foundation (Fellowship DRG-2149-13 to A.J.F).