CHK1 inhibitor induced PARylation by targeting PARG causes excessive replication and metabolic stress and overcomes chemoresistance in ovarian cancer.

Chemoresistance contributes to the majority of deaths in women with ovarian cancer (OC). Altered DNA repair and metabolic signaling is implicated in mediating therapeutic resistance. DNA damage checkpoint kinase 1 (CHK1) integrates cell cycle and DNA repair in replicating cells, and its inhibition causes replication stress, repair deficiency and cell cycle dysregulation. We observed elevated Poly-ADP-ribosylation (PAR) of proteins (PARylation) and subsequent decrease in cellular NAD+ levels in OC cells treated with the CHK1 inhibitor prexasertib, indicating activation of NAD+ dependent DNA repair enzymes poly-ADP-ribose polymerases (PARP1/2). While multiple PARP inhibitors are in clinical use in treating OC, tumor resistance to these drugs is highly imminent. We reasoned that inhibition of dePARylation by targeting Poly (ADP-ribose) glycohydrolase (PARG) would disrupt metabolic and DNA repair crosstalk to overcome chemoresistance. Although PARG inhibition (PARGi) trapped PARylation of the proteins and activated CHK1, it did not cause any significant OC cell death. However, OC cells deficient in CHK1 were hypersensitive to PARGi, suggesting a role for metabolic and DNA repair crosstalk in protection of OC cells. Correspondingly, OC cells treated with a combination of CHK1 and PARG inhibitors exhibited excessive replication stress-mediated DNA lesions, cell cycle dysregulation, and mitotic catastrophe compared to individual drugs. Interestingly, increased PARylation observed in combination treatment resulted in depletion of NAD+ levels. These decreased NAD+ levels were also paralleled with reduced aldehyde dehydrogenase (ALDH) activity, which requires NAD+ to maintain cancer stem cells. Furthermore, prexasertib and PARGi combinations exhibited synergistic cell death in OC cells, including an isogenic chemoresistant cell line and 3D organoid models of primary patient-derived OC cell lines. Collectively, our data highlight a novel crosstalk between metabolism and DNA repair involving replication stress and NAD+-dependent PARylation, and suggest a novel combination therapy of CHK1 and PARG inhibitors to overcome chemoresistance in OC.

GLI1-targeting drugs induce replication stress and homologous recombination deficiency and synergize with PARP-targeted therapies in triple negative breast cancer cells.

Triple negative breast cancer (TNBC), an aggressive and highly metastatic subtype of breast cancer. Glioma-associated oncogene 1 (GLI1) is a transcription factor and effector of the Hedgehog (Hh) signaling pathway, and is predictive of poor survival for TNBC patients. A nanostring DNA Damage Response (DDR) mRNA panel was used to identify GLI1-induced regulation of DDR genes. Western blots, immunohistochemistry and immunofluorescence were used to evaluate protein expression. Colony assays and mammosphere formation assays were utilized to assess survival of cancer cells. Flow cytometry analyses were employed to evaluate changes in the cell cycle profile, and DNA fiber assays were used to analyze alterations in replication dynamics in TNBC cells. The UALCAN portal and Ensemble programs were used for computational analysis of TCGA data. CompuSyn software was used to calculate combination index (CI) values to assess synergism in drug combination experiments. Inhibition of GLI1 in TNBC cells transcriptionally downregulate expression of FANCD2 and its foci formation, and causes a homologous recombination repair (HR) deficiency. As HR-deficient cancer cells are sensitive to PARP-targeted therapies, we evaluated a combination of the GLI1 inhibitor, GANT61, and a PARP inhibitor (olaparib) in TNBC cells. Combination of GANT61 and olaparib elevated DNA damage levels and these drug combinations caused synergistic lethality to TNBC cells. Aberrantly activated GLI1 regulates HR-mediated DNA repair by transcriptionally regulating FANCD2 to overcome chemotherapy-induced replication stress and DNA damage, and it contributes to resistance of TNBC cells to therapeutics.

Hedgehog/GLI1 Transcriptionally Regulates FANCD2 in Ovarian Tumor Cells: Its Inhibition Induces HR-Deficiency and Synergistic Lethality with PARP Inhibition.

Ovarian cancer (OC) is one of the most lethal type of cancer in women due to a lack of effective targeted therapies and high rates of treatment resistance and disease recurrence. Recently Poly (ADP-ribose) polymerase inhibitors (PARPi) have shown promise as chemotherapeutic agents; however, their efficacy is limited to a small fraction of patients with BRCA mutations. Here we show a novel function for the Hedgehog (Hh) transcription factor Glioma associated protein 1 (GLI1) in regulation of key Fanconi anemia (FA) gene, FANCD2 in OC cells. GLI1 inhibition in HR-proficient OC cells induces HR deficiency (BRCAness), replication stress and synergistic lethality when combined with PARP inhibition. Treatment of OC cells with combination of GLI1 and PARP inhibitors shows enhanced DNA damage, synergy in cytotoxicity, and strong in vivo anticancer responses.

Case Report: Intra-Tumoral Vaccinations of Quadrivalent HPV-L1 Peptide Vaccine With Topical TLR-7 Agonist Following Recurrence: Complete Resolution of HPV-HR-Associated Gynecologic Squamous Cell Carcinomas in Two Patients.

The human papilloma virus (HPV) high-risk variants (HPV-HR) such as HPV16 and HPV18 are responsible for most HPV related cancers, including anogenital and head and neck cancers. Here, we present two patients with HPV-HR-associated gynecological malignancies who, after failing radiation therapy, were treated with experimental salvage immunotherapy regimen resulting in complete, durable responses in both patients. Each patient was diagnosed with recurrent, radiation-refractory, HPV-HR positive, squamous cell carcinoma of the lower genital tract. Patient A was a 90-year-old, African American, with metastatic vulvar cancer to the right inguinal-femoral triangle and pulmonary metastases. Patient B was a 41-year-old, Caucasian, with a central-recurrence of cervix cancer. Each patient received at least two intratumoral quadrivalent HPV-L1 vaccine (Gardasil™) injections and daily topical TLR-7 agonist (imiquimod) to the tumor surface 2 weeks apart. This combination of intratumoral vaccinations and topical TLR-7 agonist produced unexpected complete resolution of disease in both patients. The importance of radiation therapy, despite being considered a treatment failure by current definitions, cannot be understated. Radiation therapy appears to have offered a therapeutic immune advantage by modifying the tumor microenvironment. This immune protocol has potential to help patients with advanced HPV-HR-related malignancies previously considered incurable.

MT2-MMP expression during early avian morphogenesis.

Membrane-type 2 matrix metalloproteinase (MT2-MMP; also called MMP15) is a membrane-bound protease that degrades extracellular matrix and activates proMMPs such as proMMP-2. MMP-2 expression in avian embryos is well documented, but it is not clear how proMMP-2 is activated during avian embryogenesis. Herein, we report that MT2-MMP mRNA is expressed in several tissues including the neural folds and epidermal ectoderm, intermediate mesoderm, pharyngeal arches, limb buds, and dermis. Several, but not all, of these tissues are known to express MMP-2. These observations suggest MT2-MMP may play a role during embryonic development not only through its own proteolytic activity but also by activating proMMP-2.

Homocysteine enhances cardiac neural crest cell attachment in vitro by increasing intracellular calcium levels.

Elevated homocysteine (Hcys) increases the risk of neurocristopathies. Previous studies show Hcys inhibits neural crest (NC) cell migration in vivo. However, the mechanisms responsible for this effect are unknown. Here, we evaluated the effect of Hcys on NC cell attachment in vitro and determined if any of the effects were due to altered Ca2+ signaling. We found Hcys enhanced NC cell attachment in a dose and substrate-dependent manner. Ionomycin mimicked the effect of Hcys while BAPTA-AM and 2-APB blocked the effect of Hcys on NC attachment. In contrast, inhibitors of plasma membrane Ca2+ channels had no effect on NC attachment. Hcys also increased the emission of the intracellular Ca2+-sensitive probe, Fluo-4. These results show Hcys alters NC attachment by triggering an increase in intracellular Ca2+ possibly by generating inositol triphosphate. Hence, the teratogenic effect ascribed to Hcys may be due to perturbation of intracellular Ca2+ signaling.

Mutant myocilin nonsecretion in vivo is not sufficient to cause glaucoma.

Glaucoma is a leading cause of blindness, affecting over 70 million people worldwide. Vision loss is the result of death of the retinal ganglion cells. The best-known risk factor for glaucoma is an elevated intraocular pressure (IOP); however, factors leading to IOP elevation are poorly understood. Mutations in the MYOC gene are an important cause of open-angle glaucoma. Over 70 MYOC mutations have been identified, and they lead to approximately 5% of all primary open-angle glaucoma cases. Nevertheless, the pathogenic mechanisms by which these mutations elevate IOP are presently unclear. Data suggest that a dominant interfering effect of misfolded mutant MYOC molecules may be pathogenic. To test this hypothesis, we have generated mice carrying a mutant allele of Myoc that is analogous to a human mutation that leads to aggressive glaucoma in patients. We show that mutant MYOC is not secreted into the aqueous humor. Instead of being secreted, mutant MYOC accumulates within the iridocorneal angle of the eye, consistent with the behavior of abnormally folded protein. Surprisingly, the accumulated mutant protein does not activate the unfolded protein response and lead to elevated intraocular pressure or glaucoma in aged mice of different strains. These data suggest that production, apparent misfolding, and nonsecretion of mutant MYOC are not, by themselves, sufficient to cause glaucoma in vivo.

Homocysteine inhibits cardiac neural crest cell formation and morphogenesis in vivo.

Elevated homocysteine increases the risk of neurocristopathies. Here, we determined whether elevating homocysteine altered the proliferation or number of chick neural crest cells that form between the midotic and third somite in vivo. Homocysteine increased the number of neural tube cells but decreased neural crest cell number. However, the sum total of cells was not different from controls. In controls, the 5-bromo-2'-deoxyuridine-labeling index was higher in newly formed neural crest cells than in their progenitors, paralleling reports showing these progenitors must pass the restriction point before undergoing epithelial-mesenchymal transition. Homocysteine decreased the labeling index of newly formed neural crest cells, suggesting that it inhibited cell cycle progression of neural crest progenitors or the S-phase entry of newly formed neural crest cells. Homocysteine also inhibited neural crest dispersal and decreased the distance they migrated from the neural tube. These results show neural crest morphogenesis is directly altered by elevated homocysteine in vivo. Developmental Dynamics 229:63-73, 2004.

The expression patterns of c-kit and Sl in chicken embryos suggest unexpected roles for these genes in somite and limb development.

We have used whole-mount in situ hybridization to investigate the patterns of c-kit and Sl expression in stage 11-22 chicken embryos. Our analysis shows that c-kit and Sl are expressed quite differently in chicken embryos compared to the reported expression patterns of these genes in embryos of other taxa. Most notably, chicken c-kit is expressed in primordial germ cells as well as in the developing somite, the apical ectodermal ridge, and in the early foregut endoderm. Sl is expressed in the lateral and intermediate mesoderm and in extraembryonic membranes. These data suggest that chicken c-kit and Sl may play novel and unexpected roles in somitogenesis, limb development, and foregut development in avian embryos.

Contribution of BRCA1 and BRCA2 to familial ovarian cancer: a gynecologic oncology group study.

OBJECTIVES: The aim of the study was to determine the prevalence of BRCA1 and BRCA2 germline mutations among ovarian cancer patients ascertained to have a family history of ovarian cancer. METHODS: Ovarian cancer patients were eligible if they had a family history of cancer that met any one of the following criteria: (1) a first-degree relative with ovarian cancer; (2) a second-degree relative with ovarian cancer plus a first-degree relative with breast cancer (diagnosed younger than 50 years of age); or (3) a first- and a second-degree relative with breast cancer (diagnosed younger than 50 years of age). The entire coding sequence of BRCA1 and exon 11 of BRCA2 were screened for germline alterations by single-strand conformation polymorphism analysis. RESULTS: Of 26 eligible patients screened for mutations, 12 had deleterious alterations, 8 in BRCA1 and 4 in BRCA2. A correlation was noted between the presence of a BRCA1 mutation and the strength of family history of breast ovarian cancer, with the likelihood of a mutation increasing with the number of affected relatives (P = 0.0002). No association was detected between the location of mutations in BRCA1 and the ratio of ovarian cancer cases relative to breast cancer (P = 0.28). CONCLUSIONS: Mutations in BRCA1 or BRCA2 are present in about 50% of ovarian cancer patients with at least one first-degree relative with disease, and in 70% of patients with two or more relatives with ovarian cancer.