<p dir="ltr">In this study, we presented the discovery of the first-in-class covalent inhibitor specific to Src homology 2 domain containing phosphatase 1 (SHP1), an overlooked cancer immunotherapy target. Through high-throughput screening, we identified a chloroacetamide fragment highly selective for SHP1. This fragment was subsequently refined to yield M029, a covalent inhibitor characterized by low-micromolar potency, heightened selectivity, enhanced stability, and improved bioavailability. Notably, M029 targets a cryptic, non-conserved cysteine residue on SHP1, thereby illuminating novel avenues for future drug development focused on SHP1. This presented study also marked the first characterization of SHP1 pharmacology inhibition <i>in vivo</i> using M029 as a tool compound. Consistent to previous genetic studies, SHP1 inhibition was observed to markedly bolster anti-tumor efficacy, primarily through the activation of CD8+ T cells and NK cells, coupled with a reduction in T cell exhaustion. While no synergistic effects were noted in conjunction with anti-PD-1 treatment, M029 as a standalone therapy showcased more favorable responses compared to anti-PD-1 therapy alone, underscoring its potential for clinical application.</p><p dir="ltr">Meanwhile, we also demonstrated the effects of targeting both protein tyrosine phosphatase 1B (PTP1B), and T cell protein tyrosine phosphatase (TC-PTP) using proteolysis targeting chimeras (PROTACs). PROTACs are heterobifunctional small molecules comprising a targeted protein ligand, an E3 ligase ligand, and a linker. By recruiting an E3 ligase to the targeted proteins, PROTACs leverages the cell's ubiquitin-proteasome machinery to achieve selective target protein degradation. In contrast to traditional occupancy-based inhibitors, event-driven PROTACs show improved efficacy by promoting target protein degradation in a catalytic mode of action and greater selectivity through the obligatory formation of the target-PROTAC-E3 ternary complex, which is essential for efficient target degradation. Through optimizing the previously reported PROTAC DU-14, we acquired a cereblon (CRBN)-based PTP1B/TC-PTP dual targeting PROTAC X1 of higher bioavailability than DU-14. X1 showed enhanced efficacy than DU-14 in multiple cell lines and manifested anti-cancer efficacy <i>in vivo</i>. Additionally, employing X1 as a tool compound, we validated the anti-cancer potential of PTP1B/TC-PTP degradation in STAT3 dependent malignancies, such as non-Hodgkin’s lymphomas. Treatments with X1 or DU-14 effectively induced tumor cell apoptosis, whereas the dual inhibitor ABBV-CLS-484 failed to produce comparable outcomes.</p>

Trinucleotide repeat (TNR) instability including repeat expansions and repeat deletions is the cause of more than 40 inherited incurable neurodegenerative diseases and cancer. TNR instability is associated with DNA damage and base excision repair (BER). In this dissertation research, we explored the mechanisms of BER-mediated TNR instability via biochemical analysis of the BER protein activities, DNA structures, protein-protein interaction, and protein-DNA interaction by reconstructing BER in vitro using synthesized oligonucleotide TNR substrates and purified human proteins. First, we evaluated a germline DNA polymerase β (pol β) polymorphic variant, pol βR137Q, in leading TNR instability-mediated cancers or neurodegenerative diseases. We find that the pol βR137Q has slightly weaker DNA synthesis activity compared to that of wild-type (WT) pol β. Because of the similar abilities between pol βR137Q and WT pol β in bypassing a template loop structure, both pol βR137Q and WT pol β induces similar amount of repeat deletion. We conclude that the slightly weaker DNA synthesis activity of pol βR137Q does not alter the TNR instability compared to that of WT pol β, suggesting that the pol βR137Q carriers do not have an altered risk in developing TNR instability-mediated human diseases. We then investigated the role of DNA synthesis activities of DNA polymerases in modulating TNR instability. We find that pol βY265C and pol ν with very weak DNA synthesis activities predominantly promote TNR deletions. We identify that the sequences of TNRs may also affect DNA synthesis and alter the outcomes of TNR instability. By inhibiting the DNA synthesis activity of pol β using a pol β inhibitor, we find that the outcome of TNR instability is shifted toward repeat deletions. The results provide the direct evidence that DNA synthesis activity of DNA polymerases can be utilized as a potential therapeutic target for treating TNR expansion diseases. Finally, we explored the role of post-translational modification (PTM) of proliferating cell nuclear antigen (PCNA) on TNR instability. We find that ubiquitinated PCNA (ub-PCNA) stimulates Fanconi associated nuclease 1 (FAN1) 5’-3’ exonucleolytic activities directly on hairpin structures, coordinating flap endonuclease 1 (FEN1) in removing difficult secondary structures, thereby suppressing TNR expansions. The results suggest a role of mono-ubiquitination of PCNA in maintaining TNR stability by regulating nucleases switching. Our results suggest enzymatic activities of DNA polymerases and nucleases and the regulation of the activities by PTM play important roles in BER-mediated TNR instability. This research provides the molecular basis for future development of new therapeutic strategies for prevention and treatment of TNR-mediated neurodegenerative diseases.

Hohe Telomeraseaktivität bedingt durch genomische TERT-Rearrangements definiert eine Gruppe an Hochrisiko-Neuroblastompatienten mit ungünstiger Prognose. Das Abzielen auf Telomerase ist ein hochpriorisierter Ansatzpunkt in der Therapie, für die es bislang keine klinisch erfolgreichen Inhibitoren gibt. Der Einsatz von epigenetisch wirksamen Histondeacetylase Inhibitoren (HDACi) stellt dabei eine interessante Therapieoption dar. In TERT-rearrangierten Neuroblastomzellen erzielte die Behandlung mit verschiedenen pan-, Klasse I oder spezifischen HDAC1/2 Inhibitoren eine Supprimierung der TERT mRNA Expression und der Telomeraseaktivität. RNA-Interferenz Studien bestätigten, dass HDAC1 und HDAC2 die TERT Expression positiv regulieren. Die transiente Überexpression von TERT zeigte einen partiellen Rescue des HDACi-bedingten anti-proliferativen Effekts. Der präventive und therapeutische Einsatz von HDACi Panobinostat verlangsamte das Xenografttumorwachstum, die TERT-Expression und Telomeraseaktivität in subkutanen NMRI-Foxn1nu/nu Mausmodellen des TERT-rearrangierten Neuroblastoms bei klinisch relevanten Dosen. Dies zeigt das translationale Potential und die klinische Durchführbarkeit der Panobinostat-Behandlung. ChIP Sequenzierung und Methylierungsanalyse zeigten keine bedeutenden Unterschiede der Histonmodifikationen und der Methylierung von CpG Dinukleotiden am TERT Lokus nach Panobinostatbehandlung. Die Inhibierung der de novo RNA Synthese zeigte, dass die Stabilität des TERT mRNA Transkripts nach Panobinostatbehandlung verringert war. Dies deutet darauf hin, dass die reduzierte Transkriptstabilität der zugrundeliegende molekulare Mechanismus ist. Zusammenfassend konnte gezeigt werden, dass die hohe Telomeraseaktivität in TERT-rearrangierten Neuroblastommodellen durch den Einsatz zugelassener HDACi supprimiert werden kann. / Telomerase activation by genomic TERT-rearrangements defines a subgroup of high-risk neuroblastomas with adverse outcome. Accordingly, telomerase activity presents a high-priority drug target with no currently available clinical inhibitors. It was assessed whether telomerase activity could be inhibited through histone deacetylase (HDAC) inhibition in models of TERT-rearranged neuroblastoma. Treatment with a panel of seven pan-, class I- or specific HDAC1/2 inhibitors suppressed TERT mRNA expression and telomerase activity in TERT-rearranged neuroblastoma cells at clinically achievable concentrations. RNA interference-based studies confirmed that HDAC1 and HDAC2 positively regulate TERT transcript levels. Enforced TERT expression partly rescued the anti-proliferative effect of HDAC inhibition indicating a causal role of TERT suppression in the HDAC inhibitormediated tumor-suppressive phenotype. Panobinostat treatment, in preventive and therapeutic settings, considerably attenuated tumor growth in subcutaneous TERT-rearranged neuroblastoma xenograft models in NMRI-Foxn1nu/nu mice and suppressed TERT transcript levels and telomerase activity at clinically relevant doses, thus demonstrating translational potential and clinical feasibility. ChIP sequencing detected no major differences in the chromatin context of the TERT locus between HDAC inhibitor-treated and control cells. Likewise, HDAC inhibition did not substantially alter the methylation profile in the TERT region. Blocking de novo RNA synthesis, however, reduced TERT mRNA transcript levels in HDAC inhibitor-treated cells, suggesting reduced TERT transcript stability as the underlying molecular mechanism. In summary, high-level telomerase activity caused by genomic rearrangements in neuroblastoma models is suppressed by treatment with clinically approved HDAC inhibitors, suggesting indirect druggability and a potential molecular rationale for therapeutic intervention.