Abstract Introduction. Zelenirstat (PCLX-001) is a small molecule inhibitor of N-myristoylation, a process that involves addition of the fatty acid myristate to over 200 proteins by two N-myristoyltransferases (NMT1 and 2). These include Src family kinases and c-Abl. Inhibition of N-myristoylation by zelenirstat leads to the degradation of non-myristoylated proteins and cancer cell death. We conducted a phase I trial to evaluate the safety, tolerability, and maximally tolerated dose (MTD) of zelenirstat in patients with refractory cancer. Methods. Differential mass spectrometry was performed in NMT1 and NMT2 CRISPR/Cas9 KOs or zelenirstat-treated HAP1 cells to identify N-myristoylation-regulated proteins. Fully consented patients with advanced solid malignancies or refractory B-cell lymphomas were administered escalating doses of zelenirstat in 28-day cycles until progression or dose-limiting toxicity (DLT). Results. Proteomic analysis of cells with genetic or pharmacological NMT inhibition was performed to gain insights into NMT substrates and function. In addition to the rapid degradation of SFKs, which disrupts the pro-survival signaling of RTKs, we identified multiple respiratory complex I proteins as the most degraded, including NDUFAF4. NMT1 KO and zelenirstat treatment disrupted complex I leading to oxidative phosphorylation (OXPHOS) inhibition, which is essential for both cancer stem cell survival and metastasis. Continuous once-daily zelenirstat at 20 mg to 210 mg was well tolerated, with no dose-limiting toxicities in 24 patients up to and including 210 mg. Gastrointestinal DLTs were observed in the 280 mg cohort, establishing 210 mg as the MTD. Oral absorption was rapid, and pharmacokinetics were suitable for once daily dosing. Seven patients had stable disease as best response, including pancreatic, ovarian and colon cancer patients; one colon cancer patient with 5 prior lines of therapy continues 210 mg beyond 11 cycles with reduction in carcinoembryonic antigen (CEA) and tumor dimensions. Kaplan-Meier analysis revealed longer progression-free survival (log-rank p=0.033) and overall survival (p=0.026) in the patients treated at 210 mg (n=7) when compared with patients treated in lower dose cohorts (n=17). Conclusion. Zelenirstat has a unique dual impact on growth signaling and OXPHOS. Having previously demonstrated pre-clinical efficacy in hematologic cancers in vitro and in vivo, we now demonstrate the therapeutic potential of zelenirstat in patients with refractory advanced solid malignancies, warranting further clinical evaluation in these indications. Citation Format: Luc Gerard Berthiaume, Erwan Beauchamp, Jay Gamma, Rony Pain, Morris A. Kostiuk, Christopher R. Cromwell, Eman W. Moussa, Olivier Julien, Basil P. Hubbard, John Kuruvilla, Laurie H. Sehn, Jennifer Spratlin, Rahima Jamal, Randeep Sangha, John R. Mackey. N-myristoylation inhibitor zelenirstat: New mechanistic insights and efficacy signals from a first in human phase I study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT194.
Abstract Two N-myristoyltransferases (NMTs) NMT1 and NMT2 catalyze the reaction. NMT1 is ubiquitous expressed and is essential for cell survival while NMT2 is more variably expressed and non-essential suggesting that their substrate specificity and activity levels differ. Historically, inhibition of myristoylation was suggested as a therapeutic anti-cancer target since NMTs expression were shown to be increased in numerous types of cancers and myristoylation was shown to be essential for proper localization and activity of some important proto-oncogenes such as Src Family Kinases (SFKs). Recently, we showed NMT2 expression is lost in numerous haematological cancer cell lines (including AML) and that these haematological cancer cell lines are exquisitely sensitive to the pan-NMT inhibitor PCLX-001. PCLX-001 recently entered human clinical trials as once daily oral therapy for relapsed/refractory B-cell Non-Hodgkin Lymphoma and advanced solid malignances. Dysregulation and oncogenic activity of SFKs occurs frequently in AML, suggesting NMT inhibition could provide therapeutic benefit in this indication. Data analysis from the TCGA transcriptome database revealed that high NMT1 and low NMT2 were associated with reduced overall and event-free survival in adult AML. Moreover, high NMT1 - but not NMT2 - expression is associated with proliferative gene sets in AML cell lines. AML cell lines treated with PCLX-001 showed a significant reduction in total protein myristoylation, reduced levels of SFK proteins and SFK phosphorylation as well as significant increases in ER stress marker BIP protein and caspase 3 cleavage. PCLX-001 induced apoptosis in AML cell lines and patient blasts at concentrations that spared a large proportion of peripheral blood lymphocytes and monocytes from healthy individuals. PCLX-001 monotherapy had dose-dependent anticancer activity in an AML MV-4-11 cell line derived xenograft (CDX) and two AML patient derived xenografts (PDXs) and produced complete remissions in subcutaneous AML CDX. In tail-vein injected PDX models, PCLX-001 treatment resulted in up to 95% reduction of human CD45+ cells in peripheral blood and bone marrow. PCLX-001 preferentially targeted AML cells inducing apoptosis and reducing leukemic burden. These findings validate NMT inhibition as a novel therapeutic strategy for AML and warrant the evaluation of PCLX-001 in clinical trials for adult AML. Citation Format: Jay Gamma, Aishwarya Iyer, Megan Yap, Zoulika Zak, Krista Vincent, Cassidy Ekstrom, Qiang Liu, Erwan Beauchamp, Lynne Postovit, Jean Wang, John R. Mackey, Naveen Pemmaraju, Gautam Borthakur, Joseph Brandwein, Luc Berthiaume. Targeting N-myristoylation for therapy of adult acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5662.
Treatment of aggressive lymphoma is toxic, expensive, and a substantial proportion of patients relapse and die. There is an urgent need for more effective treatments. Myristoylation is required for biological activity of >200 intracellular proteins. N-myristoyltransferases (NMTs) transfer the fatty acid myristate to N-terminal glycine residue; there are two isoforms, NMT1 and 2. Since they are critical to intracellular signaling, NMTs are potential anti-cancer targets. We tested a novel potent pan-NMT inhibitor, PCLX-001, in B cell lymphoma cell lines. In vitro assays included cell viability, immunoblotting, and metabolic labeling of lymphoma cell lines. Immunohistochemistry was performed on formalin fixed paraffin embedded lymphoma specimens from patients. In vivo experiments included cell line derived murine xenografts and a patient derived mouse xenograft treated with increasing concentrations of PCLX-001. PCLX-001 selectively killed lymphoma cells, while sparing normal cells in vitro and in 3 mouse xenograft models, eradicating tumors in two of these models including a patient-derived xenograft from a R-CHOP refractory lymphoma patient. While NMT2 is overexpressed in some cancers, loss of NMT2 expression is common in numerous cancers and occurs at the highest prevalence in lymphomas, where it is independently linked to a worse prognosis. This NMT2 suppression occurred through epigenetic mechanisms and may account for lymphoma sensitivity to NMT inhibition. The global myristoylation of lymphoma cell proteins, including that of the protein tyrosine kinase oncogene Src, is profoundly inhibited by PCLX-001. Loss of Src myristoylation is accompanied by loss of Src activity and may account for loss of prosurvival signals causing lymphoma cell death. Targeting NMT2 deficient B cell lymphoma with a pan-NMT inhibitor suppresses the residual NMT1 function provides a novel, selective, and effective therapeutic strategy.Citation Format: John R. Mackey, Erwan Beauchamp, Megan C. Yap, Aishwarya Iyer, Maneka A. Perinpanayagam, Krista M. Vincent, Abass M. Al-Momany, Ryan J. Heit, Jacky Y. Sim, Raymond Lai, Wei-feng Dong, Manikandan Lakshmanan, Anandhkumar Raju, Vinay Tergaonkar, Soo Yong Tan, Soon Thye Lim, Lynne M. Postovit, Kevin D. Read, David W. Gray, Paul G. Wyatt, Luc G. Berthiaume. Targeting N-myristoylation in B cell lymphomas as a therapeutic strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3046.
Transient receptor potential polycystin-3 (TRPP3) is a cation channel activated by calcium and proton and is involved in hedgehog signaling, intestinal development, and sour tasting. How TRPP3 channel function is regulated remains poorly understood. By N-terminal truncation mutations, electrophysiology, and Xenopus oocyte expression, we first identified fragment Asp-21–Ser-42 to be functionally important. We then found that deletion mutant Δ1–36 (TRPP3 missing fragment Met-1–Arg-36) has a similar function as wild-type TRPP3, whereas Δ1–38 is functionally dead, suggesting the importance of Val-37 or Cys-38. Further studies found that Cys-38, but not Val-37, is functionally critical. Cys-38 is a predicted site of palmitoylation, and indeed TRPP3 channel activity was inhibited by palmitoylation inhibitor 2-bromopalmitate and rescued by palmitoylation substrate palmitic acid. The TRPP3 N terminus (TRPP3NT, Met-1–Leu-95) localized along the plasma membrane of HEK293 cells but stayed in the cytoplasm with 2-bromopalmitate treatment or C38A mutation, indicating that TRPP3NT anchors to the surface membrane through palmitoylation at Cys-38. By acyl-biotin exchange assays, we showed that TRPP3, but not mutant C38A, is indeed palmitoylated. When putative phosphorylation sites near Cys-38 were mutated to Asp or Glu to mimic phosphorylation, only T39D and T39E reduced TRPP3 function. Furthermore, TRPP3NT displayed double bands in which the upper band was abolished by λ phosphatase treatment or T39A mutation. However, palmitoylation at Cys-38 and phosphorylation at Thr-39 independently regulated TRPP3 channel function, in contrast to previous reports about correlated palmitoylation with a proximate phosphorylation. Palmitoylation at Cys-38 represents a novel mechanism of functional regulation for TRPP3.
The palmitoylation of calnexin serves to enrich calnexin on the mitochondria-associated membrane (MAM). Given a lack of information on the significance of this finding, we have investigated how this endoplasmic reticulum (ER)-internal sorting signal affects the functions of calnexin. Our results demonstrate that palmitoylated calnexin interacts with sarcoendoplasmic reticulum (SR) calcium transport ATPase (SERCA) 2b and that this interaction determines ER calcium content and the regulation of ER-mitochondria calcium crosstalk. In contrast, non-palmitoylated calnexin interacts with the oxidoreductase ERp57 and performs its well-known function in quality control. Interestingly, our results also show that calnexin palmitoylation is an ER stress-dependent mechanism. Following a short term ER stress, calnexin quickly becomes less palmitoylated, which shifts its function from the regulation of calcium signaling towards chaperoning and quality control of known substrates. These changes also correlate with a preferential distribution of calnexin to the MAM under resting conditions or the rough ER and ER quality control compartment (ERQC) following ER stress. Our results have therefore identified the switch that assigns calnexin either to calcium signaling or to protein chaperoning.
Abstract Myristoylation is required for biological activity of >200 intracellular proteins. N-myristoyltransferases (NMTs) transfer the fatty acid myristate to N-terminal glycine residue; there are two isoforms, NMT1 and 2. In acute myeloid leukemia (AML), upregulation of Lyn and Src, important myristoylated proteins, contribute to cell survival and proliferation. The specific roles of NMT1 and NMT2 are unknown in this context. The relationships among NMT1/NMT2 expression and acute AML patient outcomes were studied using RNA-sequencing and microarray cohorts with over 350 patients. We found that high NMT1 and low NMT2 expression were associated with reduced overall and event-free survival in adult AML, which was independent of other prognostic markers on multivariate analysis. High NMT1, but not NMT2, expression was associated with proliferative gene sets in AML cell lines, indicating potential for distinct isozyme substrates. Given these results, we examined NMT1 and NMT2 levels in AML cell lines and AML patient blast cells using Western blotting and flow cytometry. We determined that NMT2 expression varied greatly among patients, but was markedly reduced in most cases, while NMT1 expression was relatively preserved. A potent small molecule NMT inhibitor, PCLX-001, preferentially induced apoptosis and reduced viability in NMT2-deficient AML cell lines cultured in vitro compared with normal lymphocytes and peripheral blood mononuclear cells. PCLX-001 also killed freshly isolated human AML blasts ex vivo with an IC50 of ~170nM regardless of their mutational background. In a murine AML xenograft model, subcutaneously delivered PCLX-001 monotherapy demonstrated dose-dependent anticancer activity and produced complete remissions after five daily 50 mg/kg doses. NMT expression provides independent prognostic information to refine existing clinical stratification, and NMT inhibition is a promising novel therapeutic strategy for AML. Citation Format: John R. Mackey, Aishwarya Iyer, Megan C. Yap, Zoulika Zak, Krista Vincent, Erwan Beauchamp, Lynne M. Postovit, Joseph Brandwein, Luc G. Berthiaume. Examination of NMT1 and NMT2 as independent prognostic markers and novel drug targets in adult acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3043.
<p>Supplementary Figure S4: Expression of NMT1 and NMT2 as it associates with AML risk factors. Patients were separated based on mutational status of commonly used prognostic genes and compared based on expression of NMT1 (A) and NMT2 (B). No significant differences were found in displayed genes. Patients were separated into risk categories based on ELN2022 guidelines or TCGA-LAML molecular risk and expression of NMT1 and NMT2 evaluated (C). No significant differences were found.</p>
<p>Figure S7: SFKs respond similarly to zelenirstat. AML cell lines were pre-treated with 1µM zelenirstat for 48 hours, then stimulated with 100 ng/mL SCF & FL. Levels of indicated Src-family kinases were analyzed by western blot.</p>
