Search

Supplementary Figure 7. Targeting of TRIM37 suppresses TNBC metastasis.

The majority of clinical deaths in patients with triple-negative breast cancer (TNBC) are due to chemoresistance and aggressive metastases, with high prevalence in younger women of African ethnicity. Although tumorigenic drivers are numerous and varied, the drivers of metastatic transition remain largely unknown. Here, we uncovered a molecular dependence of TNBC tumors on the TRIM37 network, which enables tumor cells to resist chemotherapeutic as well as metastatic stress. TRIM37-directed histone H2A monoubiquitination enforces changes in DNA repair that rendered TP53-mutant TNBC cells resistant to chemotherapy. Chemotherapeutic drugs triggered a positive feedback loop via ATM/E2F1/STAT signaling, amplifying the TRIM37 network in chemoresistant cancer cells. High expression of TRIM37 induced transcriptomic changes characteristic of a metastatic phenotype, and inhibition of TRIM37 substantially reduced the in vivo propensity of TNBC cells. Selective delivery of TRIM37-specific antisense oligonucleotides using antifolate receptor 1–conjugated nanoparticles in combination with chemotherapy suppressed lung metastasis in spontaneous metastatic murine models. Collectively, these findings establish TRIM37 as a clinically relevant target with opportunities for therapeutic intervention.Significance:TRIM37 drives aggressive TNBC biology by promoting resistance to chemotherapy and inducing a prometastatic transcriptional program; inhibition of TRIM37 increases chemotherapy efficacy and reduces metastasis risk in patients with TNBC.

Table I: List of TRIM37-associated DSB repair and cell proliferation genes expressed in non-TNBC, TNBC and normal tissue obtained from METABRIC patient cohort.

Supplementary Figure 4. Chemotherapy activates ATM/E2F1/STAT axis to upregulate TRIM37 in TNBC.

Supplementary Methods and Material

The majority of clinical deaths in patients with triple-negative breast cancer (TNBC) are due to chemoresistance and aggressive metastases, with high prevalence in younger women of African ethnicity. Although tumorigenic drivers are numerous and varied, the drivers of metastatic transition remain largely unknown. Here, we uncovered a molecular dependence of TNBC tumors on the TRIM37 network, which enables tumor cells to resist chemotherapeutic as well as metastatic stress. TRIM37-directed histone H2A monoubiquitination enforces changes in DNA repair that rendered TP53-mutant TNBC cells resistant to chemotherapy. Chemotherapeutic drugs triggered a positive feedback loop via ATM/E2F1/STAT signaling, amplifying the TRIM37 network in chemoresistant cancer cells. High expression of TRIM37 induced transcriptomic changes characteristic of a metastatic phenotype, and inhibition of TRIM37 substantially reduced the in vivo propensity of TNBC cells. Selective delivery of TRIM37-specific antisense oligonucleotides using antifolate receptor 1–conjugated nanoparticles in combination with chemotherapy suppressed lung metastasis in spontaneous metastatic murine models. Collectively, these findings establish TRIM37 as a clinically relevant target with opportunities for therapeutic intervention.Significance:TRIM37 drives aggressive TNBC biology by promoting resistance to chemotherapy and inducing a prometastatic transcriptional program; inhibition of TRIM37 increases chemotherapy efficacy and reduces metastasis risk in patients with TNBC.

The novel and highly pathogenic coronavirus (SARS-CoV-2) remains a public health threat worldwide. SARS-CoV-2 enters human host lung cells via its spike protein binding to angiotensin-converting enzyme 2 (ACE2) in a process critical dependent on host protease-mediated fusion event. Thus, effective targeted therapies blocking the first step of viral fusion and cellular entry remains a critical unmet medical need to overcome disease pathology. Here we engineered and describe an antibody-based novel and targeted plug-and-play strategy, which directly competes with the proteolytic activation function of SAR-CoV-2 spike protein. The described strategy involves the engineering of furin substrate residues in IgG1 Fc-extended flexible region of spike targeting antibody. Our results with spike receptor-binding domain (RBD) targeting CR3022 antibody support blockade of the viral function using proof of concept ACE2 overexpressing cells. Our study reveals analytical, safe, and selective mechanistic insights for SARS-CoV-2 therapeutic design and is broadly applicable to the future coronaviridae family members (including mutant variants) exploiting the host protease system for cellular entry.

Lack of effective immune infiltration represents a significant barrier to immunotherapy in solid tumors. Thus, solid tumor‐enriched death receptor‐5 (DR5) activating antibodies, which generates tumor debulking by extrinsic apoptotic cytotoxicity, remains a crucial alternate therapeutic strategy. Over past few decades, many DR5 antibodies moved to clinical trials after successfully controlling tumors in immunodeficient tumor xenografts. However, DR5 antibodies failed to significantly improve survival in phase‐II trials, leading in efforts to generate second generation of DR5 agonists to supersize apoptotic cytotoxicity in tumors. Here we have discovered that clinical DR5 antibodies activate an unexpected immunosuppressive PD‐L1 stabilization pathway, which potentially had contributed to their limited success in clinics. The DR5 agonist stimulated caspase‐8 signaling not only activates ROCK1 but also undermines proteasome function, both of which contributes to increased PD‐L1 stability on tumor cell surface. Targeting DR5‐ROCK1‐PD‐L1 axis markedly increases immune effector T‐cell function, promotes tumor regression, and improves overall survival in animal models. These insights have identified a potential clinically viable combinatorial strategy to revive solid cancer immunotherapy using death receptor agonism., DR5 agonists moved to clinical trials after successfully controlling tumors in immunodeficient xenograft models. By stabling introducing human DR5 extracellular domain into murine tumor cells, this study identifies PD‐L1 pathway as a contributing factor for potential immune evasion in solid tumors.

Therapeutic antibodies targeting ovarian cancer (OvCa)-enriched receptors have largely been disappointing due to limited tumor specific antibody-dependent cellular cytotoxicity (ADCC). Here we report a symbiotic approach that is highly selective and superior compared to investigational clinical antibodies. This Bispecific-Anchored Cytotoxicity-Activator (BaCa) antibody is rationally designed to instigate “cis” and “trans” cytotoxicity by combining specificities against folate receptor alpha-1 (FOLR1) and death receptor 5 (DR5). Whereas the in vivo agonist DR5 signaling requires FcγRIIB interaction, the FOLR1 anchor functions as a primary clustering point to retain and maintain a high-level of tumor-specific apoptosis. The presented proof of concept study strategically makes use of a tumor-cell enriched anchor receptor for agonist death-receptor targeting to potentially generate a clinically viable strategy for OvCa.

Alkylating agents induce cytotoxic DNA base adducts. In this work, we provide evidence to suggest, for the first time, that Saccharomyces cerevisiae Tpa1 protein is involved in DNA alkylation repair. Little is known about Tpa1 as a repair protein beyond the initial observation from a high-throughput analysis indicating that deletion of TPA1 causes methyl methane sulfonate sensitivity in S. cerevisiae. Using purified Tpa1, we demonstrate that Tpa1 repairs both single- and double-stranded methylated DNA. Tpa1 is a member of the Fe(II) and 2-oxoglutarate-dependent dioxygenase family, and we show that mutation of the amino acid residues involved in cofactor binding abolishes the Tpa1 DNA repair activity. Deletion of TPA1 along with the base excision repair pathway DNA glycosylase MAG1 renders the tpa1Δmag1Δ double mutant highly susceptible to methylation-induced toxicity. We further demonstrate that the trans-lesion synthesis DNA polymerase Polζ (REV3) plays a key role in tolerating DNA methyl-base lesions and that tpa1Δmag1revΔ3 triple mutant is extremely susceptible to methylation-induced toxicity. Our results indicate a synergism between the base excision repair pathway and direct alkylation repair by Tpa1 in S. cerevisiae. We conclude that Tpa1 is a hitherto unidentified DNA repair protein in yeast and that it plays a crucial role in reverting alkylated DNA base lesions and cytotoxicity.

The Escherichia coli AlkB protein is a 2-oxoglutarate/Fe(II)-dependent demethylase that repairs alkylated single stranded and double stranded DNA. Immunoaffinity chromatography coupled with mass spectrometry identified RecA, a key factor in homologous recombination, as an AlkB-associated protein. The interaction between AlkB and RecA was validated by yeast two-hybrid assay; size-exclusion chromatography and standard pull down experiment and was shown to be direct and mediated by the N-terminal domain of RecA. RecA binding results AlkB-RecA heterodimer formation and RecA-AlkB repairs alkylated DNA with higher efficiency than AlkB alone.

Ubiquitin C-terminal hydrolase-1 (UCH-L1) is a specific neuronal endoprotease that cleaves the specific peptide bond between ubiquitin molecules. UCH-L1 is released in serum and cerebrospinal fluid after severe brain injury and is considered to be an important biomarker of brain injury. A common polymorphism of UCH-L1 (S18Y) is also linked to a reduced risk of Parkinson's disease. In addition to its function in neuronal tissues, UCH-L1 may also play a part in the progression of certain non-neuronal cancers. UCH-L1 is highly expressed in primary lung tumors and colo-rectal cancers, suggesting a role in tumorigenesis. We report here the development of a sensitive and accurate UCH-L1 assay based on the surface plasmon resonance (SPR) absorbance of gold nanoparticles. We created a unique UCH-L1 substrate containing a ubiquitin molecule with two terminal thiol groups. This UCH-L1 substrate interacted with gold nanoparticles via the terminal thiol groups and induced clustering of the nanoparticles, which was detected by SPR absorbance at 650 nm. UCH-L1 proteolytically cleaved the substrate and the clustered gold nanoparticles were dispersed and could be detected by a shift in the SPR absorbance to 530 nm. This change in absorbance was proportional to the concentration of UCH-L1 and can be used for the quantification of functional UCH-L1. The currently available fluorescence-based UCH-L1 assay is affected by a high background signal and a poor detection limit, especially in the presence of serum. The assay reported here can detect concentrations of UCH-L1 as low as 20 ng ml−1 (0.8 nM) and the presence of serum had no effect on the detection limit. This assay could be adapted for the rapid determination of the severity of brain injury and could also be applied to high-throughput screening of inhibitors of UCH-L1 enzymatic activity in Parkinson's disease and cancer., This work was funded by the Department of Biotechnology (DBT), Government of India (Project no. 102/IFD/SAN/634/2013–2014; DT 09-05-2013).