MV1035 Overcomes Temozolomide Resistance in Patient-Derived Glioblastoma Stem Cell Lines

Glioblastoma (GBM, grade IV glioma) represents the most aggressive brain tumor and patients with GBM have a poor prognosis. Until now surgical resection followed by radiotherapy and temozolomide (TMZ) treatment represents the standard strategy for GBM. We showed that the imidazobenzoxazin-5-thione MV1035 is able to significantly reduce GBM U87-MG cells migration and invasiveness through inhibition of the RNA demethylase ALKBH5. In this work, we focus on the DNA repair protein ALKBH2, a further MV1035 target resulting from SPILLO-PBSS proteome-wide scale in silico analysis. Our data demonstrate that MV1035 inhibits the activity of ALKBH2, known to be involved in GBM TMZ resistance. MV1035 was used on both U87-MG and two patient-derived (PD) glioma stem cells (GSCs): in combination with TMZ, it has a significant synergistic effect in reducing cell viability and sphere formation. Moreover, MV1035 induces a reduction in MGMT expression in PD-GSCs cell lines most likely through a mechanism that acts on MGMT promoter methylation. Taken together our data show that MV1035 could act as an inhibitor potentially helpful to overcome TMZ resistance and able to reduce GBM migration and invasiveness.

Rapid genome editing by CRISPR-Cas9-POLD3 fusion

Precision CRISPR gene editing relies on the cellular homology-directed DNA repair (HDR) to introduce custom DNA sequences to target sites. The HDR editing efficiency varies between cell types and genomic sites, and the sources of this variation are incompletely understood. Here, we have studied the effect of 450 DNA repair protein - Cas9 fusions on CRISPR genome editing outcomes. We find the majority of fusions to improve precision genome editing only modestly in a locus- and cell-type specific manner. We identify Cas9-POLD3 fusion that enhances editing by speeding up the initiation of DNA repair. We conclude that while DNA repair protein fusions to Cas9 can improve HDR CRISPR editing, most need to be optimized to the cell type and genomic site, highlighting the diversity of factors contributing to locus-specific genome editing outcomes.

CBIO-15. LOSS OF WILLIAMS SYNDROME TRANSCRIPTION FACTOR (WSTF) LEADS TO IMPROVED TEMOZOLOMIDE SENSITIVITY IN HUMAN GLIOBLASTOMA CELLS IRRESPECTIVE OF MGMT EXPRESSION

BACKGROUND The prognosis for glioblastoma multiforme (GBM) patients is poor with a median survival of approximately 15 months. The DNA repair protein O 6-methylguanine-DNA methyltransferase (MGMT) counteracts the effects of temozolomide (TMZ) chemotherapy and is thus associated with poor outcome in GBM patients. Williams Syndrome Transcription Factor (WSTF) has been suggested to regulate the DNA damage response pathway (DDR) in both an indirect (through chromatin remodeling) and direct manner (by phosphorylating H2AX at Tyr142). However, whether WSTF has any role in the development of resistance against chemotherapy through its functions in the DDR in GBMs, is so far unknown. In this study, we investigated whether a loss of WSTF sensitizes different MGMT-proficient and -deficient GBM cell lines to TMZ treatment. METHODS We generated WSTF knockout clones from both MGMT-proficient (LN18, T98G) and -deficient GBM cell lines (U-251) using CRISPR/Cas9 gene-editing technology with lentiviral vectors. The PCR-based screening results combined with the T7 endonuclease mismatch assay for bi-allelic monoclonal knockouts were verified via sequencing and immunoblotting to identify candidate knockout clones. Colony formation assays were performed to determine the survival ability in response to TMZ treatment. Statistical analysis was performed using two-way ANOVA. RESULTS WSTF knockout clones showed a significant decrease in colony formation after TMZ-treatment compared to the corresponding control groups (non-target single guide RNA) (LN18: Clone 59 vs control: p= 0.0456, T98G: All three studied clones vs control: p< 0.0001, U-251: Clone 7/35.1/70.2 vs control: p< 0.0001/p= 0.0107/p= 0.0119). CONCLUSION WSTF is an important factor in both MGMT de- and proficient GBM cell lines for response against TMZ chemotherapy. The loss of WSTF leads to a significantly increased TMZ sensitivity in clinically relevant concentrations for all the studied cell lines. Ongoing studies are investigating the underlying mechanisms and potential alterations in the DDR pathway caused by WSTF loss.

DDRE-29. EGFR INHIBITION DOWNREGULATES MGMT AND SENSITIZES GBM CELLS TO TMZ

Glioblastoma (GBM) is a highly malignant type of adult brain tumor with a poor prognosis. Temozolomide (TMZ), a DNA alkylating agent, has been widely used as an effective first-line chemotherapeutic agent for the treatment of GBM patients. The efficacy of TMZ in GBM depends on the absence of the DNA repair protein MGMT which reverses the DNA damage induced by TMZ. The MGMT promoter is hypermethylated in about 45% of GBMs, resulting in lack of MGMT expression and increased responsiveness to TMZ. TMZ is less effective in MGMT unmethylated GBMs. We propose that EGFR inhibition downregulates MGMT and sensitizes glioma cells to TMZ and a combination of pretreatment with erlotinib followed by TMZ could be a useful therapeutic approach in MGMT expressing GBMs. As our experimental model, we used multiple MGMT unmethylated lines from the Mayo Clinic patient derived xenografts (PDXs) panel. Our data demonstrate that exposure of cells to erlotinib for 48h results in downregulation of MGMT at the mRNA and protein level. Additionally, EGFR inhibition activates the AP-1 transcription factor, and overexpression of AP-1 components Fos and Jun results in decreased MGMT expression in TMZ resistant PDXs, suggesting that AP-1 acts as a transcriptional repressor of MGMT. We further identified that the mice implanted with TMZ resistant PDXs pretreated with afatinib followed by TMZ treatment survived longer compared to those treated with TMZ alone. Thus, the use of EGFR inhibition may enhance the sensitivity of MGMT unmethylated GBMs to TMZ.

SARS–CoV–2 Spike Impairs DNA Damage Repair and Inhibits V(D)J Recombination In Vitro

Severe acute respiratory syndrome coronavirus 2 (SARS–CoV–2) has led to the coronavirus disease 2019 (COVID–19) pandemic, severely affecting public health and the global economy. Adaptive immunity plays a crucial role in fighting against SARS–CoV–2 infection and directly influences the clinical outcomes of patients. Clinical studies have indicated that patients with severe COVID–19 exhibit delayed and weak adaptive immune responses; however, the mechanism by which SARS–CoV–2 impedes adaptive immunity remains unclear. Here, by using an in vitro cell line, we report that the SARS–CoV–2 spike protein significantly inhibits DNA damage repair, which is required for effective V(D)J recombination in adaptive immunity. Mechanistically, we found that the spike protein localizes in the nucleus and inhibits DNA damage repair by impeding key DNA repair protein BRCA1 and 53BP1 recruitment to the damage site. Our findings reveal a potential molecular mechanism by which the spike protein might impede adaptive immunity and underscore the potential side effects of full-length spike-based vaccines.

Innovative 3D proteome-wide scale identification of ALKBH5 target for MV1035 small molecule able to reduce migration and invasiveness in U87 glioblastoma cell lines by SPILLO-PBSS

The innovative in silico technologies developed at SPILLOproject,1 e.g., the SPILLO potential binding sites searcher (SPILLO-PBSS) software,2,3 allow to identify targets and off-targets of any small molecule on a multiple-organism proteomewide scale, and to perform an accurate multilevel cross-organism transferability analysis (MCOTA) aimed at rationalising animal testing. SPILLO-PBSS has been successfully used in several research projects, such as a study in which a compound (MV1035) was found to reduce migration and invasiveness in U87 glioblastoma (GBM) cell lines: the human structural proteome was analyzed and the RNA demethylase ALKBH5 has been identified as a target responsible for the observed effects (target experimentally validated). Another top-ranked target identified by SPILLO-PBSS, the DNA repair protein AlkB homolog 2 (ALKBH2), abundantly expressed in GBM cell lines, resulted particularly interesting for its pivotal role in the onset of resistance to Temozolomide (TMZ), the standard firstline treatment for GBM.2

Molecular Functions of Hydrogen Sulfide in Cancer

Hydrogen sulfide (H2S) is a gasotransmitter that exerts a multitude of functions in both physiologic and pathophysiologic processes. H2S-synthesizing enzymes are increased in a variety of human malignancies, including colon, prostate, breast, renal, urothelial, ovarian, oral squamous cell, and thyroid cancers. In cancer, H2S promotes tumor growth, cellular and mitochondrial bioenergetics, migration, invasion, angiogenesis, tumor blood flow, metastasis, epithelia–mesenchymal transition, DNA repair, protein sulfhydration, and chemotherapy resistance Additionally, in some malignancies, increased H2S-synthesizing enzyme expression correlates with a worse prognosis and a higher tumor stage. Here we review the role of H2S in cancer, with an emphasis on the molecular mechanisms by which H2S promotes cancer development, progression, dedifferentiation, and metastasis.

P15.02 Loss of Williams Syndrome Transcription Factor (WSTF) leads to improved temozolomide sensitivity in human glioblastoma cells irrespective of MGMT expression

BACKGROUND The prognosis for newly diagnosed adult glioblastoma multiforme (GBM) patients is poor even after standard therapy with a median survival of approximately 14–15 months. The DNA repair protein O 6 -methylguanine-DNA methyltransferase (MGMT) efficiently counteracts formation of the most lethal DNA adducts by temozolomide (TMZ) chemotherapy, and is thus associated with poor outcome in GBM patients. Williams Syndrome Transcription Factor (WSTF) has previously been suggested to regulate the DNA damage response pathway (DDR) in both an indirect (through chromatin remodeling together with SMARCA5 in the WICH complex) and direct manner (by phosphorylating H2AX at Tyr142). However, whether WSTF has any role in the development of resistance against chemotherapy through its ability to regulate the DDR in GBMs, is so far not known. In this study, we investigated whether loss of WSTF sensitizes different MGMT-proficient and -deficient GBM cell lines to TMZ treatment. MATERIAL AND METHODS We generated WSTF knockout clones from both MGMT-proficient (LN18, T98G) and -deficient GBM cell lines (U-251) using CRISPR/Cas9 gene-editing technology with lentiviral vectors. The PCR-based screening results combined with the T7 endonuclease mismatch assay for bi-allelic monoclonal knockouts were verified via sequencing and immunoblotting to identify candidate knockout clones. For each cell line, three knockout clones were chosen for further investigation. Colony formation assays were performed to determine the survival ability in response to TMZ treatment. Statistical analysis was performed using two-way ANOVA. RESULTS WSTF knockout clones showed a significant decrease in colony formation after TMZ-treatment compared to the corresponding WSTF-expressing control groups (non-target single guide RNA) (LN18: Clone 59 vs control: p= 0.0456, T98G: All three studied clones vs control: p <0.0001, U-251: Clone 7/35.1/70.2 vs control: p <0.0001/p= 0.0107/p= 0.0119). Furthermore, two out of three clones of T98G and U-251 (T98G Clone 13 and 128 vs control, p <0.0001, U-251 Clone 7 vs control, p= 0.0062; clone 70.2, p= 0.0052) showed significantly reduced plating efficiency compared to control cells. CONCLUSION WSTF is an important factor in both MGMT de- and proficient GBM cell lines for response against TMZ chemotherapy. The loss of WSTF leads to a significantly increased TMZ sensitivity in clinically relevant concentrations for all the studied cell lines. Ongoing studies are investigating the underlying mechanisms and potential alterations in the DDR pathway caused by WSTF loss.