Targeting N-myristoylation for therapy of B-cell lymphomas

Abstract

Myristoylation, the N-terminal modification of proteins with the fatty acid myristate, is critical for membrane targeting and cell signaling. Because cancer cells often have increased N-myristoyltransferase (NMT) expression, NMTs were proposed as anti-cancer targets. To systematically investigate this, we performed robotic cancer cell line screens and discovered a marked sensitivity of hematological cancer cell lines, including B-cell lymphomas, to the potent pan-NMT inhibitor PCLX-001. PCLX-001 treatment impacts the global myristoylation of lymphoma cell proteins and inhibits early B-cell receptor (BCR) signaling events critical for survival. In addition to abrogating myristoylation of Src family kinases, PCLX-001 also promotes their degradation and, unexpectedly, that of numerous non-myristoylated BCR effectors including c-Myc, NFκB and P-ERK, leading to cancer cell death in vitro and in xenograft models. Because some treated lymphoma patients experience relapse and die, targeting B-cell lymphomas with a NMT inhibitor potentially provides an additional much needed treatment option for lymphoma.

Conflict of interest statement

As co-founders of Pacylex Pharmaceuticals Inc. (www.pacylex.com), which owns the rights to patent applications PCT/2012/000696 and PCT/2013/050821, L.G.B., J.R.M., E.B., M.A.P., J.M.G., and M.Y. declare potential competing interests. To minimize these, cell line screens and animal xenograft studies were performed at arms length of the Pacylex co-founders. All the other authors declare no competing interests.

Figures

Fig. 1. PCLX-001 selectively kills hematological cancer cell lines in comparison to cancer cell lines of other origins.

Percentage of maximum growth inhibition of various cell lines following 96 h treatment with 1.2 μM PCLX-001 as determined using a Horizon cell line screen (a, b), or following 72 h treatment with 1 μM of PCLX-001 using a OncolinesTM cell line screen (c, d). Cell lines are arranged according to tumor cell type. Cross-hatched zone represents cytotoxic effect. Hematological cancer cell lines are depicted in gray while all other types of cancer cell lines are depicted in white. Corresponding violin graphs compare the average PCLX-001-mediated growth inhibition on hematological cancer cell lines to cancer cell lines of other origins combined as calculated from the Horizon (b) and OncolinesTM (d) cell screens (Unpaired t test, two-tailed P < 0.0001). Quartiles are separated by dotted lines. Error bars represent standard deviation within each group. Normalized cell viability curves of immortalized lymphocyte (IM9, VDS), BL (BL2, Ramos, BJAB), and DLBCL (DOHH2, WSU-DLCL2, SU-DHL-10) cell lines treated with 0.001–5 μM of PCLX-001 for 96 h as determined by CellTiter Blue Viability Assay (e). Corresponding histograms of absolute IC50 (and SD) values calculated from a log(inhibitor) vs response (three parameters) equation cell viability curves plotted in e (f). ***Indicates a significant difference (P ≤ 0.001) in IC50 between IM9 cells and all other cell lines tested (Ordinary one-way Anova, Tukey’s multiple comparisons test, P < 0.0001). Normalized proliferation of IM9 (g) and BL2 (h) cells reated with 0–5 μM of PCLX-001 for 96 h as determined by cell count. Values are mean ± s.e.m. of three independent experiments. Source data are provided as a Source Data file.

Fig. 2. PCLX-001 selectively inhibits myristoylation in vitro and induces apoptosis in lymphoma cell lines.

Click chemistry was used on alkyne-myristate labeled cell lysates to determine overall protein myristoylation levels in: BL2 cells (a) and IM9 cells (b) treated for 1 h with 0.01–1.0 µM PCLX-001, myristoylation levels of a WT-Src-EGFP construct expressed in COS-7 cells (c) and, myristoylation of immunoprecipitated endogenous pp60-Src in IM9 cells following 1 h treatment with 1.0–10 µM of PCLX-001 (d). Fluorescence micrographs of COS-7 cells transfected with a WT-Src-EGFP (left), non-myristoylatable G2A-Src-EGFP mutant (center), and a WT-Src-EGFP construct treated with 10 μM PCLX-001 for 24 h (right) (e). Scale bars are equal to 10 μm. Endogenous Src protein levels in IM9 and BL2 cells treated with 1 μM PCLX-001 for 0–5 days measured by Western blotting (f). Western blotting of cleaved PARP-1 and cleaved caspase-3 in IM9, BL2, and Ramos cell lysates following 72 h incubation with 0–1.0 µM PCLX-001 (composite gels) (g). All data shown are representative of at least three independent experiments. GAPDH serves as loading control. Source data are provided as a Source Data file.

Fig. 3. PCLX-001 treatment results in SFK instability and degradation by the proteasome in lymphoma cell lines.

Western blot for total Src and Lyn proteins in immortalized lymphocyte (IM9, VDS), BL (BL2, Ramos, BJAB), and DLBCL (DOHH2, WSU-DLCL2, SU-DHL-10) cell line lysates following 24–48 h of treatment with 0.1 µM or 1.0 μM PCLX-001 (a). After BCR ligation with anti-IgM, western blot for total Src, Lyn, Hck, Lck, Mcl-1, total phospho-tyrosine (PY99) and pan phosphorylated-SFK (P-SFK) protein levels in BL2 treated for 24–48 h with 1 µM PCLX-001 in the presence or absence of 10 µM of the proteasome inhibitor MG132 for the last 6 h (b). GAPDH serves as a loading control. All western blots shown are representative of three independent experiments. Source data are provided as a Source Data file.

Fig. 4. PCLX-001 treatment attenuates BCR downstream signaling events in BL2 lymphoma cells.

Western blot of BL2 cells treated for 48 h with 0.1 µM or 1.0 μM of dasatinib, ibrutinib or PCLX-001 to detect total tyrosine phosphorylation (P-Tyr), Lyn, Lyn phosphorylated on tyrosine 396 or 507, BTK, and BTK phosphorylated on tyrosines 223 or 551 (a), HGAL, SYK, phosphorylated SYK (P-SYK) (b) or ERK, phosphorylated ERK (P-ERK), NFκB, c-Myc, CREB, Arf-1, BIP, and PARP-1 (c). Western blots are representative of at least three independent experiments. GAPDH serves as a loading control. BL2 cells were activated with 25 μg/ml F(ab’)2 anti-human IgM for 2 min and processed for western blotting. All western blots shown are representative of three independent experiments. Source data are provided as a Source Data file.

Fig. 5. Model depicting proposed PCLX-001 mechanism of action in B cell lymphoma.

a Upon BCR activation, first the myristoylated SFK Lyn is recruited to the lipid raft domains of the plasma membrane containing the BCR, dephosphorylated Lyn at Y507 leads to its activation and autophosphorylation at Y396. This leads to the phosphorylation and activation of BTK at Y551 and Y223. Second, myristoylated HGAL is also recruited to the plasma membrane and phosphorylated thereby enhancing BCR signaling by stimulating SYK, BTK and the release of Ca++ ions from the endoplasmic reticulum via the inositol-3-phosphate ion channel receptor (IP3R). Altogether these early signaling events lead to transcription activation by c-Myc, P-ERK, NFκB, and CREB. b The NMT inhibitor PCLX-001 prevents the myristoylation of Lyn-SFK (as well as other SFKs not shown in this model), HGAL and Arf1 thereby impeding the proper membrane targeting and function of these proteins. PCLX-001 treatment impedes calcium homeostasis by reducing the BCR mediated Ca++ release from the ER and increasing basal Ca++ levels in cells in addition to promote the degradation of both myristoylated (Lyn, HGAL, Arf1) and, surprisingly, non-myristoylated proteins (NFκB, P-ERK, c-Myc and CREB), some via the ubiquitin-proteasome pathway thereby further abrogating downstream BCR signaling and increasing ER stress leading to apoptosis and cell death.

Fig. 6. PCLX-001 selectively kills hematological cancer cells relative to benign lymphocytes in comparison to dasatinib and ibrutinib.

Cell viability curves of BL2 (solid lines) and IM9 cells (dotted lines) treated for 48 h (a) or 96 h (b) with 0.001–5 µM dasatinib, ibrutinib, or PCLX-001(2way Anova, Tukey’s multiple comparisons test, P < 0.0001) . Normalized cell viability of immortalized lymphocyte (IM9, VDS), BL (BL2, Ramos, BJAB), and DLBCL (DOHH2, WSU-DLCL2, SU-DHL-10) cell lines treated with 0.1 µM or 1.0 μM of dasatinib, ibrutinib or PCLX-001 for 48 h (c) and 96 h (d). Cell viability for all experiments was measured using Calcein assay and is an average of three independent experiments. (Ordinary one-way Anova, Dunnett’s multiple comparisons test) Errors bars depict s.e.m. Source data are provided as a Source Data file.

Fig. 7. PCLX-001 treatment reduces tumor volumes and leads to complete tumor regression in B-cell lymphoma xenograft models.

Dose–response curves for murine subcutaneous xenografts derived from cell lines measuring the size of DOHH2 (a) and BL2 (b) tumors as a function of time. Error bars represent the standard deviation of average tumor volumes (n = 10 per group). Average total NMT specific activity assessed as previously described in BL2 tumor samples from mice treated with PCLX-001, doxorubicin, or vehicle alone at the indicated doses. Tumor extracted from mice treated with 60 mg/kg/day have reduced NMT specific activity as compared to vehicle (paired t-test, P = 0.0425). Error bars represent s.e.m. (c). Dose–response curve for the murine xenograft derived from patient DLBCL3. Datapoints represent average tumor volumes in all surviving animals. Error bars represent the standard deviation in the average tumor volumes (d). ***Indicates a significant difference in response rate between animals which received 20 mg/kg/day and 50 mg/kg/day of PCLX-001 (P < 0.0001, n = 8 per group). Representative tumors from mice with patient-derived DLBCL3 xenografts (e). Representative IHC staining for cleaved caspase-3 (f) and Ki-67 (g) in the above DLBCL3 patient xenograft tumor samples. Scale bars equal to 100 μm. Source data are provided as a Source Data file.