Purpose SIK2 is a centrosome kinase required for mitotic spindle formation and a potential target for ovarian cancer therapy. Here we examine the effects of a novel small molecule SIK2 inhibitor, ARN-3236, on sensitivity to paclitaxel in ovarian cancer. Experimental Design SIK2 expression was determined in ovarian cancer tissue samples and cell lines. ARN-3236 was tested for its efficiency to inhibit growth and enhance paclitaxel sensitivity in cultures and xenografts of ovarian cancer cell lines. SIK2 siRNA and ARN-3236 were compared for their ability to produce nuclear-centrosome dissociation, inhibit centrosome splitting, block mitotic progression, induce tetraploidy, trigger apoptotic cell death and reduce AKT/survivin signaling. Results SIK2 is overexpressed in approximately 30% of high grade serous ovarian cancers. ARN-3236 inhibited growth of 10 ovarian cancer cell lines at an IC50 of 0.8 to 2.6 μM, where the IC50 of ARN-3236 was inversely correlated with endogenous SIK2 expression (Pearson’s r = −0.642, P = 0.03). ARN-3236 enhanced sensitivity to paclitaxel in 8 of 10 cell lines, as well as in SKOv3ip (P = 0.028) and OVCAR8 xenografts. In at least three cell lines a synergistic interaction was observed. ARN-3236 uncoupled the centrosome from the nucleus in interphase, blocked centrosome separation in mitosis, caused prometaphase arrest and induced apoptotic cell death and tetraploidy. ARN-3236 also inhibited AKT phosphorylation and attenuated survivin expression. Conclusions ARN-3236 is the first orally available inhibitor of SIK2 to be evaluated against ovarian cancer in preclinical models and shows promise in inhibiting ovarian cancer growth and enhancing paclitaxel chemosensitivity.

BACKGROUND: Poly(adenosine diphosphate ribose) polymerase (PARP) inhibitors exhibit promising activity against ovarian cancers, but their efficacy can be limited by acquired drug resistance. This study explores the role of autophagy in regulating the sensitivity of ovarian cancer cells to PARP inhibitors. METHODS: Induction of autophagy was detected by punctate LC3 fluorescence staining, LC3I to LC3II conversion on Western blot analysis, and electron microscopy. Enhanced growth inhibition and apoptosis were observed when PARP inhibitors were used with hydroxychloroquine, chloroquine (CQ), or LYS05 to block the hydrolysis of proteins and lipids in autophagosomes or with small interfering RNA against ATG5 or ATG7 to prevent the formation of autophagosomes. The preclinical efficacy of the combination of CQ and olaparib was evaluated with a patient-derived xenograft (PDX) and the OVCAR8 human ovarian cancer cell line. RESULTS: Four PARP inhibitors (olaparib, niraparib, rucaparib, and talazoparib) induced autophagy in a panel of ovarian cancer cells. Inhibition of autophagy with CQ enhanced the sensitivity of ovarian cancer cells to PARP inhibitors. In vivo, olaparib and CQ produced additive growth inhibition in OVCAR8 xenografts and a PDX. Olaparib inhibited PARP activity, and this led to increased reactive oxygen species (ROS) and an accumulation of γ-H2AX. Inhibition of autophagy also increased ROS and γ-H2AX and enhanced the effect of olaparib on both entities. Treatment with olaparib increased phosphorylation of ATM and PTEN while decreasing the phosphorylation of AKT and mTOR and inducing autophagy. CONCLUSIONS: PARP inhibitor-induced autophagy provides an adaptive mechanism of resistance to PARP inhibitors in cancer cells with wild-type BRCA, and a combination of PARP inhibitors with CQ or other autophagy inhibitors could improve outcomes for patients with ovarian cancer. Cancer 2020;126:894-907.

BackgroundAutophagy induction can increase or decrease anticancer drug efficacy. Anticancer drug-induced autophagy induction is poorly characterized in osteosarcoma (OS). In this study, we investigated the impact of autophagy inhibition on camptothecin (CPT)-induced cytotoxicity in OS.MethodsAutophagy-inhibited DLM8 and K7M3 metastatic murine OS cell lines were generated by infection with lentiviral shRNA directed against the essential autophagy protein ATG5. Knockdown of ATG5 protein expression and inhibition of autophagy was confirmed by immunoblot of ATG5 and LC3II proteins, respectively. Metabolic activity was determined by MTT assay and cell viability was determined by trypan blue exclusion. Acridine orange staining and immunoblotting for LC3II protein expression were used to determine autophagy induction. Oxidative stress was assessed by staining cells with HE and DCFH-DA followed by flow cytometry analysis. Mitochondrial membrane potential was determined by staining cells with TMRE followed by flow cytometry analysis. Immunoblotting was used to detect caspase activation, Parp cleavage and p53 phosphorylation.ResultsAutophagy inhibition caused a greater deficit in metabolic activity and cell growth in K7M3 cells compared to DLM8 cells. K7M3 cells exhibited higher basal autophagy levels than DLM8 cells and non-transformed murine MCT3 osteoblasts. Autophagy inhibition did not affect CPT-induced DNA damage. Autophagy inhibition decreased CPT-induced cell death in DLM8 cells while increasing CPT-induced cell death in K7M3 cells. Autophagy inhibition reduced CPT-induced mitochondrial damage and CPT-induced caspase activation in DLM8 cells. Buthionine sulfoximine (BSO)-induced cell death was greater in autophagy-competent DLM8 cells and was reversed by antioxidant pretreatment. Camptothecin-induced and BSO-induced autophagy induction was also reversed by antioxidant pretreatment. Significantly, autophagy inhibition not only reduced CPT-induced oxidative stress but also reduced basal oxidative stress.ConclusionsThe results of this study indicate that autophagy inhibition can have an opposing effect on CPT-induced cytotoxicity within OS. The cytoprotective mechanism of autophagy inhibition observed in DLM8 cells involves reduced CPT-induced oxidative stress and not reduced DNA damage. Our results also reveal the novel finding that knockdown of ATG5 protein reduces both basal oxidative stress and drug-induced oxidative stress.

Poly(ADP-ribose) polymerase inhibitors (PARP inhibitors) have had an increasing role in the treatment of ovarian and breast cancers. PARP inhibitors are selectively active in cells with homologous recombination DNA repair deficiency caused by mutations in BRCA1/2 and other DNA repair pathway genes. Cancers with homologous recombination DNA repair proficiency respond poorly to PARP inhibitors. Cancers that initially respond to PARP inhibitors eventually develop drug resistance. We have identified salt-inducible kinase 2 (SIK2) inhibitors, ARN3236 and ARN3261, which decreased DNA double-strand break (DSB) repair functions and produced synthetic lethality with multiple PARP inhibitors in both homologous recombination DNA repair deficiency and proficiency cancer cells. SIK2 is required for centrosome splitting and PI3K activation and regulates cancer cell proliferation, metastasis, and sensitivity to chemotherapy. Here, we showed that SIK2 inhibitors sensitized ovarian and triple-negative breast cancer (TNBC) cells and xenografts to PARP inhibitors. SIK2 inhibitors decreased PARP enzyme activity and phosphorylation of class-IIa histone deacetylases (HDAC4/5/7). Furthermore, SIK2 inhibitors abolished class-IIa HDAC4/5/7–associated transcriptional activity of myocyte enhancer factor-2D (MEF2D), decreasing MEF2D binding to regulatory regions with high chromatin accessibility in FANCD2 , EXO1 , and XRCC4 genes, resulting in repression of their functions in the DNA DSB repair pathway. The combination of PARP inhibitors and SIK2 inhibitors provides a therapeutic strategy to enhance PARP inhibitor sensitivity for ovarian cancer and TNBC.

The presence of the Philadelphia chromosome in patients with acute lymphoblastic leukemia (Ph+ALL) is a negative prognostic indicator. Tyrosine kinase inhibitors (TKI) that target BCR/ABL, such as imatinib, have improved treatment of Ph+ALL and are generally incorporated into induction regimens. This approach has improved clinical responses, but molecular remissions are seen in less than 50% of patients leaving few treatment options in the event of relapse. Thus, identification of additional targets for therapeutic intervention has potential to improve outcomes for Ph+ALL. The human epidermal growth factor receptor 2 (ErbB2) is expressed in ∼30% of B-ALLs, and numerous small molecule inhibitors are available to prevent its activation. We analyzed a cohort of 129 ALL patient samples using reverse phase protein array (RPPA) with ErbB2 and phospho-ErbB2 antibodies and found that activity of ErbB2 was elevated in 56% of Ph+ALL as compared to just 4.8% of Ph−ALL. In two human Ph+ALL cell lines, inhibition of ErbB kinase activity with canertinib resulted in a dose-dependent decrease in the phosphorylation of an ErbB kinase signaling target p70S6-kinase T389 (by 60% in Z119 and 39% in Z181 cells at 3 µM). Downstream, phosphorylation of S6-kinase was also diminished in both cell lines in a dose-dependent manner (by 91% in both cell lines at 3 µM). Canertinib treatment increased expression of the pro-apoptotic protein Bim by as much as 144% in Z119 cells and 49% in Z181 cells, and further produced caspase-3 activation and consequent apoptotic cell death. Both canertinib and the FDA-approved ErbB1/2-directed TKI lapatinib abrogated proliferation and increased sensitivity to BCR/ABL-directed TKIs at clinically relevant doses. Our results suggest that ErbB signaling is an additional molecular target in Ph+ALL and encourage the development of clinical strategies combining ErbB and BCR/ABL kinase inhibitors for this subset of ALL patients.

Chemotherapy-induced autophagy is a proposed mechanism of chemoresistance and potential therapeutic target in osteosarcoma. We evaluated heat shock protein 27 (HSP27) and autophagy-related proteins as predictors of pathologic treatment response and prognostic markers among osteosarcoma patients who received standard chemotherapy. We analyzed 394 tumor specimens (pre-treatment, post-treatment, and metastases) from 260 osteosarcoma patients by immunohistochemistry for cytoplasmic light chain 3B (LC3B)-positive puncta, sequestosome 1 (SQSTM1), high mobility group box 1 (HMGB1), and HSP27 expression. The staining percentage and intensity for each marker were scored and the extent to which marker expression was correlated with pathologic response, relapse-free survival (RFS), and overall survival (OS) was assessed. LCB3 puncta in post-treatment primary tumors (50%) and metastases (67%) was significantly higher than in pre-treatment biopsy specimens (30%; = 0.023 and<0.001). Among 215 patients with localized osteosarcoma, both pre-treatment [multivariate hazard ratio (HR), 26.7; 95% confidence interval (CI), 1.47-484; = 0.026] and post-treatment HSP27 expression (multivariate HR, 1.85; 95% CI, 1.03-3.33; = 0.039) were associated with worse OS. Lack of LC3B puncta at resection was an independent poor prognostic marker in both univariate (HR, 1.78; 95% CI, 1.05-3.03; = 0.034) and multivariate models (HR, 1.75; 95% CI, 1.01-3.04; = 0.045). Patients with LC3B/HSP27 tumors at resection had the best 10-year OS (75%) whereas patients with LC3B/HSP27 tumors had the worst 10-year survival (25%). Neither HSP27 expression nor the presence of LCB3 puncta was correlated with pathologic treatment response. Our findings establish HSP27 expression and LC3B puncta as independent prognostic markers in osteosarcoma patients receiving standard chemotherapy and support further investigation into strategies targeting HSP27 or modulating autophagy in osteosarcoma treatment. .

Fas-associated protein with death domain (FADD) was first identified for its role in linking death receptors to the apoptotic signaling pathway with subsequent cell death. Later studies reported non-apoptotic functions for FADD in normal cells and cancer cells. Non-apoptotic functions for FADD in osteosarcoma (OS) have not been reported. In this study, FADD protein expression was knocked down in human CCHOSD, LM7, and SaOS2 OS cell lines followed by assessment of sensitivity to TNFα-or TRAIL-induced cell death. Knock down of FADD significantly increased TNFα-induced cell death in LM7 and SaOS2 cell lines. The mode of TNFα-induced cell death was apoptosis and not necroptosis. Inhibition of nuclear factor kappa B (NFκB) in wildtype cells increased TNFα-induced cell death to similar levels observed in FADD knockdown cells, suggesting a role for FADD in NFκB pro-survival cell signaling. In addition, knock down of FADD increased SMAC mimetic-mediated TNFα-induced cell death in all cell lines studied. The results of this study indicate that FADD has a pro-survival function in OS following TNFα treatment that involves NFκB signaling. The results also indicate that the pro-survival function of FADD is associated with XIAP activity.

Autophagy is a catabolic process involved in cellular homeostasis. Autophagy is increased above homeostatic levels by chemotherapy, and this can either promote or inhibit tumor growth. We previously demonstrated that aerosol gemcitabine (GCB) has a therapeutic effect against osteosarcoma (OS) lung metastases. However, some tumor cells failed to respond to the treatment and persisted as isolated lung metastasis. Here, we examined the mechanisms underlying the dual role of chemotherapy-induced autophagy in OS and sought to identify biomarkers to predict OS response to treatment. In this study, we demonstrate that treatment of various OS cells with GCB induced autophagy. We also showed that GCB reduces the phosphorylation of AKT, mTOR and p70S6K and that GCB-induced autophagy in OS can lead to either cell survival or cell death. Blocking autophagy enhanced the sensitivity of LM7 OS cells and decreased the sensitivity of CCH-OS-D and K7M3 OS cells to GCB. Using a kinase array, we also demonstrated that differences in the phosphorylated heat shock protein 27 (p-HSP27) expression in the various OS cell lines after treatment with GCB, correlates to whether chemotherapy-induced autophagy will lead to increase or decrease OS cells sensitivity to therapy. Increased p-HSP27 was associated with increased sensitivity to anticancer drug treatment when autophagy is inhibited. The results of this study reveal a dual role of autophagy in OS cells sensitivity to chemotherapy and suggest that p-HSP27 could represent a predictive biomarker of whether combination therapy with autophagy modulators and chemotherapeutic drugs will be beneficial for OS patients.