Structure-Based Design of a Potent and Selective YTHDC1 Ligand.

N6-Adenosine methylation (m6A) is a prevalent post-transcriptional modification of mRNA, with YTHDC1 being the reader protein responsible for recognizing this modification in the cell nucleus. Here, we present a protein structure-based medicinal chemistry campaign that resulted in the YTHDC1 inhibitor 40, which shows an equilibrium dissociation constant (Kd) of 49 nM. The crystal structure of the complex (1.6 Å resolution) validated the design. Compound 40 is selective against the cytoplasmic m6A-RNA readers YTHDF1-3 and YTHDC2 and shows antiproliferative activity against the acute myeloid leukemia (AML) cell lines THP-1, MOLM-13, and NOMO-1. For the series of compounds that culminated into ligand 40, the good correlation between the affinity in the biochemical assay and antiproliferative activity in the THP-1 cell line provides evidence of YTHDC1 target engagement in the cell. The binding to YTHDC1 in the cell is further supported by the cellular thermal shift assay. Thus, ligand 40 is a tool compound for studying the role of YTHDC1 in AML.

Small-Molecule Inhibitors of the m7G-RNA Writer METTL1.

We discovered the first inhibitors of the m7G-RNA writer METTL1 by high-throughput docking and an enzymatic assay based on luminescence. Eleven compounds, which belong to three different chemotypes, show inhibitory activity in the range 40-300 µM. Two adenine derivatives identified by docking have very favorable ligand efficiency of 0.34 and 0.31 kcal/mol per non-hydrogen atom, respectively. Molecular dynamics simulations provide evidence that the inhibitors compete with the binding of the cosubstrate S-adenosyl methionine to METTL1. We also present a soakable crystal form that was used to determine the structure of the complex of METTL1 with sinefungin at a resolution of 1.85 Å.

Proteolysis Targeting Chimera Degraders of the METTL3-14 m6A-RNA Methyltransferase.

Methylation of adenine N6 (m6A) is the most frequent RNA modification. On mRNA, it is catalyzed by the METTL3-14 heterodimer complex, which plays a key role in acute myeloid leukemia (AML) and other types of blood cancers and solid tumors. Here, we disclose the first proteolysis targeting chimeras (PROTACs) for an epitranscriptomics protein. For designing the PROTACs, we made use of the crystal structure of the complex of METTL3-14 with a potent and selective small-molecule inhibitor (called UZH2). The optimization of the linker started from a desfluoro precursor of UZH2 whose synthesis is more efficient than that of UZH2. The first nine PROTAC molecules featured PEG- or alkyl-based linkers, but only the latter showed cell penetration. With this information in hand, we synthesized 26 PROTACs based on UZH2 and alkyl linkers of different lengths and rigidity. The formation of the ternary complex was validated by a FRET-based biochemical assay and an in vitro ubiquitination assay. The PROTACs 14, 20, 22, 24, and 30, featuring different linker types and lengths, showed 50% or higher degradation of METTL3 and/or METTL14 measured by Western blot in MOLM-13 cells. They also showed substantial degradation on three other AML cell lines and prostate cancer cell line PC3.

Therapy-Induced Senescent/Polyploid Cancer Cells Undergo Atypical Divisions Associated with Altered Expression of Meiosis, Spermatogenesis and EMT Genes.

Upon anticancer treatment, cancer cells can undergo cellular senescence, i.e., the temporal arrest of cell division, accompanied by polyploidization and subsequent amitotic divisions, giving rise to mitotically dividing progeny. In this study, we sought to further characterize the cells undergoing senescence/polyploidization and their propensity for atypical divisions. We used p53-wild type MCF-7 cells treated with irinotecan (IRI), which we have previously shown undergo senescence/polyploidization. The propensity of cells to divide was measured by a BrdU incorporation assay, Ki67 protein level (cell cycle marker) and a time-lapse technique. Advanced electron microscopy-based cell visualization and bioinformatics for gene transcription analysis were also used. We found that after IRI-treatment of MCF-7 cells, the DNA replication and Ki67 level decreased temporally. Eventually, polyploid cells divided by budding. With the use of transmission electron microscopy, we showed the presence of mononuclear small cells inside senescent/polyploid ones. A comparison of the transcriptome of senescent cells at day three with day eight (when cells just start to escape senescence) revealed an altered expression of gene sets related to meiotic cell cycles, spermatogenesis and epithelial-mesenchymal transition. Although chemotherapy (DNA damage)-induced senescence is indispensable for temporary proliferation arrest of cancer cells, this response can be followed by their polyploidization and reprogramming, leading to more fit offspring.

Chemotherapy of HER2- and MDM2-Enriched Breast Cancer Subtypes Induces Homologous Recombination DNA Repair and Chemoresistance.

Analyzing the TCGA breast cancer database, we discovered that patients with the HER2 cancer subtype and overexpression of MDM2 exhibited decreased post-treatment survival. Inhibition of MDM2 expression in the SKBR3 cell line (HER2 subtype) diminished the survival of cancer cells treated with doxorubicin, etoposide, and camptothecin. Moreover, we demonstrated that inhibition of MDM2 expression diminished DNA repair by homologous recombination (HR) and sensitized SKBR3 cells to a PARP inhibitor, olaparib. In H1299 (TP53-/-) cells treated with neocarzinostatin (NCS), overexpression of MDM2 WT or E3-dead MDM2 C478S variant stimulated the NCS-dependent phosphorylation of ATM, NBN, and BRCA1, proteins involved in HR DNA repair. However, overexpression of chaperone-dead MDM2 K454A variant diminished phosphorylation of these proteins as well as the HR DNA repair. Moreover, we demonstrated that, upon NCS treatment, MDM2 K454A interacted with NBN more efficiently than MDM2 WT and that MDM2 WT was degraded more efficiently than MDM2 K454A. Using a proliferation assay, we showed that overexpression of MDM2 WT, but not MDM2 K454A, led to acquisition of resistance to NCS. The presented results indicate that, following chemotherapy, MDM2 WT was released from MDM2-NBN complex and efficiently degraded, hence allowing extensive HR DNA repair leading to the acquisition of chemoresistance by cancer cells.

Chromatin-Directed Proteomics Identifies ZNF84 as a p53-Independent Regulator of p21 in Genotoxic Stress Response.

The p21WAF1/Cip1 protein, encoded by CDKN1A, plays a vital role in senescence, and its transcriptional control by the tumour suppressor p53 is well-established. However, p21 can also be regulated in a p53-independent manner, by mechanisms that still remain less understood. We aimed to expand the knowledge about p53-independent senescence by looking for novel players involved in CDKN1A regulation. We used a chromatin-directed proteomic approach and identified ZNF84 as a novel regulator of p21 in various p53-deficient cell lines treated with cytostatic dose of doxorubicin. Knock-down of ZNF84, an as-yet un-characterized protein, inhibited p21 gene and protein expression in response to doxorubicin, it attenuated senescence and was associated with enhanced proliferation, indicating that ZNF84-deficiency can favor senescence bypass. ZNF84 deficiency was also associated with transcriptomic changes in genes governing various cancer-relevant processes e.g., mitosis. In cells with ZNF84 knock-down we discovered significantly lower level of H2AX Ser139 phosphorylation (γH2AX), which is triggered by DNA double strand breaks. Intriguingly, we observed a reverse correlation between the level of ZNF84 expression and survival rate of colon cancer patients. In conclusion, ZNF84, whose function was previously not recognized, was identified here as a critical p53-independent regulator of senescence, opening possibilities for its targeting in novel therapies of p53-null cancers.

Novel role for the testis-enriched HSPA2 protein in regulating epidermal keratinocyte differentiation.

HSPA2, a poorly characterized member of the HSPA (HSP70) chaperone family, is a testis-enriched protein involved in male germ cell differentiation. Previously, we revealed that HSPA2 is present in human stratified epithelia, including epidermis, however the contribution of this protein to epithelial biology remained unknown. Here, we show for the first time that HSPA2 is expressed in basal epidermal keratinocytes, albeit not in keratinocytes exhibiting features attributed to primitive undifferentiated progenitors, and participates in the keratinocyte differentiation process. We found that HSPA2 is dispensable for protection of HaCaT keratinocytes against heat shock-induced cytotoxicity. We also shown that lentiviral-mediated shRNA silencing of HSPA2 expression in HaCaT cells caused a set of phenotypic changes characteristic for keratinocytes committed to terminal differentiation such as reduced clonogenic potential, impaired adhesiveness and increased basal and confluency-induced expression of differentiation markers. Moreover, the fraction of undifferentiated cells that rapidly adhered to collagen IV was less numerous in HSPA2-deficient cells than in the control. In a 3D reconstructed human epidermis model, HSPA2 deficiency resulted in accelerated development of a filaggrin-positive layer. Collectively, our results clearly show a link between HSPA2 expression and maintenance of keratinocytes in an undifferentiated state in the basal layer of the epidermis. It seems that HSPA2 could retain keratinocytes from premature entry into the terminal differentiation process. Overall, HSPA2 appears to be necessary for controlling development of properly stratified epidermis and thus for maintenance of skin homeostasis.

Molecular chaperones in the acquisition of cancer cell chemoresistance with mutated TP53 and MDM2 up-regulation.

Utilizing the TCGA PANCAN12 dataset we discovered that cancer patients with mutations in TP53 tumor suppressor and overexpression of MDM2 oncogene exhibited decreased survival post treatment. Interestingly, in the case of breast cancer patients, this phenomenon correlated with high expression level of several molecular chaperones belonging to the HSPA, DNAJB and HSPC families. To verify the hypothesis that such a genetic background may promote chaperone-mediated chemoresistance, we employed breast and lung cancer cell lines that constitutively overexpressed heat shock proteins and have shown that HSPA1A/HSP70 and DNAJB1/HSP40 facilitated the binding of mutated p53 to the TAp73α protein. This chaperone-mediated mutated p53-TAp73α complex induced chemoresistance to DNA damaging reagents, like Cisplatin, Doxorubicin, Etoposide or Camptothecin. Importantly, when the MDM2 oncogene was overexpressed, heat shock proteins were displaced and a stable multiprotein complex comprising of mutated p53-TAp73α-MDM2 was formed, additionally amplifying cancer cells chemoresistance. Our findings demonstrate that molecular chaperones aid cancer cells in surviving the cytotoxic effect of chemotherapeutics and may have therapeutic implications.