MGL S3 Chimeric Enzyme Drives Apoptotic Death of EGFR-Dependent Cancer Cells through ERK Downregulation.

Methionine dependence of malignant cells is one of the cancer hallmarks. It is well described that methionine deprivation drives cancer cells death, both in vitro and in vivo. Methionine gamma-lyase (MGL) isolated from different species or obtained by genetic engineering can be used for effective methionine depletion. In this work, we show that MGL S3, a genetically engineered protein comprised of MGL from Clostridium sporogenesis fused to epidermal growth factor (EGF)-like peptide, reduces, in vitro, the number of cancer cells of four different origins-neuroblastoma, lung, breast, and colon cancer. We reveal that MGL S3 is more toxic for neuroblastoma SH-SY5Y and lung cancer H1299 cells compared to MGL tetani, and causes cell death by the induction of apoptosis. In addition, the observed death of cells treated with MGL S3 is accompanied by the prominent downregulation of ERK activity. By the analysis of transcriptomic data of more than 1500 cancer cell lines and patient samples, we show that the high expression of four genes from the methionine metabolism pathway (AHCY, CBS, DNMT3A, and MTAP) is associated with poor prognosis for breast cancer and neuroblastoma patients. Additionally, cells of these origins are characterized by a high correlation between EGFR dependency and DNMT3A/CBS expression. Finally, we demonstrate the ability of MGL S3 to enhance the sensitivity of H1299 cells to EGFR inhibition with gefitinib.

Identification of cell type-specific correlations between ERK activity and cell viability upon treatment with ERK1/2 inhibitors.

Increased MAPK signaling is a hallmark of various cancers and is a central regulator of cell survival. Direct ERK1/2 inhibition is considered a promising approach to avoid ERK1/2 reactivation caused by upstream kinases BRAF, MEK1/2, and KRAS, as well as by receptor tyrosine kinase inhibitors, but the dynamics and selectivity of ERK1/2 inhibitors are much less studied compared with BRAF or MEK inhibitors. Using ERK1/2 and downstream kinase ELK1 reporter cell lines of lung cancer (H1299; NRASQ61K), colon cancer (HCT-116; KRASG13D), neuroblastoma (SH-SY5Y), and leukemia (U937), we examined the relationship between ERK inhibition and drug-induced toxicity for five ERK inhibitors: SCH772984, ravoxertinib, LY3214996, ulixertinib, and VX-11e, as well as one MEK inhibitor, PD0325901. Comparing cell viability and ERK inhibition revealed different ERK dependencies for these cell lines. We identify several drugs, such as SCH772984 and VX-11e, which induce excessive toxicity not directly related to ERK1/2 inhibition in specific cell lines. We also show that PD0325901, LY3214996, and ulixertinib are prone to ERK1/2 reactivation over time. We distinguished two types of ERK1/2 reactivation: the first could be reversed by adding a fresh dose of inhibitors, while the second persists even after additional treatments. We also showed that cells that became resistant to the MEK1/2 inhibitor PD0325901 due to ERK1/2 reactivation remained sensitive to ERK1/2 inhibitor ulixertinib. Our data indicate that correlation of ERK inhibition with drug-induced toxicity in multiple cell lines may help to find more selective and effective ERK1/2 inhibitors.

Subtype of Neuroblastoma Cells with High KIT Expression Are Dependent on KIT and Its Knockdown Induces Compensatory Activation of Pro-Survival Signaling.

Neuroblastoma (NB) is a pediatric cancer with high clinical and molecular heterogeneity, and patients with high-risk tumors have limited treatment options. Receptor tyrosine kinase KIT has been identified as a potential marker of high-risk NB and a promising target for NB treatment. We investigated 19,145 tumor RNA expression and molecular pathway activation profiles for 20 cancer types and detected relatively high levels of KIT expression in NB. Increased KIT expression was associated with activation of cell survival pathways, downregulated apoptosis induction, and cell cycle checkpoint control pathways. KIT knockdown with shRNA encoded by lentiviral vectors in SH-SY5Y cells led to reduced cell proliferation and apoptosis induction up to 50%. Our data suggest that apoptosis induction was caused by mitotic catastrophe, and there was a 2-fold decrease in percentage of G2-M cell cycle phase after KIT knockdown. We found that KIT knockdown in NB cells leads to strong upregulation of other pro-survival growth factor signaling cascades such as EPO, NGF, IL-6, and IGF-1 pathways. NGF, IGF-1 and EPO were able to increase cell proliferation in KIT-depleted cells in an ERK1/2-dependent manner. Overall, we show that KIT is a promising therapeutic target in NB, although such therapy efficiency could be impeded by growth factor signaling activation.

Enhancer RNA AL928768.3 from the IGH Locus Regulates MYC Expression and Controls the Proliferation and Chemoresistance of Burkitt Lymphoma Cells with IGH/MYC Translocation.

Chromosomal rearrangements leading to the relocation of proto-oncogenes into transcription-active regions are found in various types of tumors. In particular, the transfer of proto-oncogenes to the locus of heavy chains of immunoglobulins (IGH) is frequently observed in B-lymphomas. The increased expression of the MYC proto-oncogene due to IGH/MYC translocation is detected in approximately 85% of Burkitt lymphoma cases. The regulatory mechanisms affecting the oncogenes upon translocation include non-coding enhancer RNAs (eRNAs). We conducted a search for the eRNAs that may affect MYC transcription in the case of IGH/MYC translocation in Burkitt lymphoma, looking for potentially oncogenic eRNAs located at the IGH locus and predominantly expressed in B cells. Overexpression and knockdown of our primary candidate eRNA AL928768.3 led to the corresponding changes in the expression of MYC proto-oncogene in Burkitt lymphoma cells. Furthermore, we demonstrated that AL928768.3 knockdown decreased lymphoma cell proliferation and resistance to chemotherapy. Significant effects were observed only in cell lines bearing IGH/MYC abnormality but not in B-cell lines without this translocation nor primary B-cells. Our results indicate that AL928768.3 plays an important role in the development of Burkitt's lymphoma and suggest it and similar, yet undiscovered eRNAs as potential tissue-specific targets for cancer treatment.