Anti-MYCN (aa 329-359) polyclonal antibody

Loss-of-function mutations of EZH2, the enzymatic component of PRC2, have been associated with poor outcome and chemotherapy resistance in T-cell acute lymphoblastic leukemia (T-ALL). Using isogenic T-ALL cells, with and without CRISPR/Cas9-induced EZH2-inactivating mutations, we performed a cell-based synthetic lethal drug screen. EZH2-deficient cells exhibited increased sensitivity to structurally diverse inhibitors of CHK1, an interaction that could be validated genetically. Furthermore, small-molecule inhibition of CHK1 had efficacy in delaying tumor progression in isogenic EZH2-deficient, but not EZH2 wild-type, T-ALL cells in vivo, as well as in a primary cell model of PRC2-mutant ALL. Mechanistically, EZH2 deficiency resulted in a gene-expression signature of immature T-ALL cells, marked transcriptional upregulation of MYCN, increased replication stress, and enhanced dependency on CHK1 for cell survival. Finally, we demonstrate this phenotype is mediated through derepression of a distal PRC2-regulated MYCN enhancer. In conclusion, we highlight a novel and clinically exploitable pathway in high-risk EZH2-mutated T-ALL. SIGNIFICANCE: Loss-of-function mutations of PRC2 genes are associated with chemotherapy resistance in T-ALL, yet no specific therapy for this aggressive subtype is currently clinically available. Our work demonstrates that loss of EZH2 activity leads to MYCN-driven replication stress, resulting in increased sensitivity to CHK1 inhibition, a finding with immediate clinical relevance.

Neuroblastoma, the most common extracranial solid tumor in children, accounts for nearly 8% of childhood cancers in the United States. It is a disease with pronounced clinical and biological heterogeneities. The amplification of MYCN, whose key tumorigenic functions include the promotion of proliferation, facilitation of the cell's entry into the S phase, and prevention of cells from leaving the cell cycle, correlates with poor prognosis. Patients with a high proliferation index disease have low survival rates. Neuroblastoma is one of the most radioresponsive of all human tumors. To exploit this radiosensitivity, radioactive guanidine (R)-(-)-5-[I-125]iodo-3 '-O-[2-(epsilon-guanidinohexanoyl)-2-phenylacetyl]-2 '-deoxyuridine (9, GPAID) was designed. This compound enters neuroblastoma cells much like metaiodobenzylguanidine (MIBG). Additionally, it cotargets DNA of proliferating cells, an attribute especially advantageous in the treatment of MYCN-amplified tumors. GPAID was synthesized from the trimethylstannyl precursor with an average yield of >90% at the no-carrier-added specific activities. The norepinephrine transporter-aided delivery of GPAID to neuroblastoma cells was established in the competitive uptake studies with nonradioactive MIBG. The intracellular processing and DNA targeting properties were confirmed in the subcellular distribution experiments. Studies in a mouse model of neuroblastoma demonstrated the therapeutic potential of GPAID. The tin precursor of GPAID can be used to prepare compounds radiolabeled with single-photon emission computed tomography (SPECT)- and positron-emission tomography (PET)-compatible radionuclides. Accordingly, these reagents can function as theranostics useful in the individualized and comprehensive treatment strategies comprising treatment planning and the assessment of tumor responses as well as the targeted molecular radiotherapy employing treatment doses derived from the imaging data.