Elimination of dormant, autophagic ovarian cancer cells and xenografts through enhanced sensitivity to anaplastic lymphoma kinase inhibition.

BACKGROUND: Poor outcomes for patients with ovarian cancer relate to dormant, drug-resistant cancer cells that survive after primary surgery and chemotherapy. Ovarian cancer (OvCa) cells persist in poorly vascularized scars on the peritoneal surface and depend on autophagy to survive nutrient deprivation. The authors have sought drugs that target autophagic cancer cells selectively to eliminate residual disease. METHODS: By using unbiased small-interfering RNA (siRNA) screens, the authors observed that knockdown of anaplastic lymphoma kinase (ALK) reduced the survival of autophagic OvCa cells. Small-molecule ALK inhibitors were evaluated for their selective toxicity against autophagic OvCa cell lines and xenografts. Autophagy was induced by reexpression of GTP-binding protein Di-Ras3 (DIRAS3) or serum starvation and was evaluated with Western blot analysis, fluorescence imaging, and transmission electron microscopy. Signaling pathways required for crizotinib-induced apoptosis of autophagic cells were explored with flow cytometric analysis, Western blot analysis, short-hairpin RNA knockdown of autophagic proteins, and small-molecule inhibitors of STAT3 and BCL-2. RESULTS: Induction of autophagy by reexpression of DIRAS3 or serum starvation in multiple OvCa cell lines significantly reduced the 50% inhibitory concentration of crizotinib and other ALK inhibitors. In 2 human OvCa xenograft models, the DIRAS3-expressing tumors treated with crizotinib had significantly decreased tumor burden and long-term survival in 67% to 79% of mice. Crizotinib treatment of autophagic cancer cells further enhanced autophagy and induced autophagy-mediated apoptosis by decreasing phosphorylated STAT3 and BCL-2 signaling. CONCLUSIONS: Crizotinib may eliminate dormant, autophagic, drug-resistant OvCa cells that remain after conventional cytoreductive surgery and combination chemotherapy. A clinical trial of ALK inhibitors as maintenance therapy after second-look operations should be seriously considered.

Poly(adenosine diphosphate ribose) polymerase inhibitors induce autophagy-mediated drug resistance in ovarian cancer cells, xenografts, and patient-derived xenograft models.

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.

Crizotinib enhances PARP inhibitor efficacy in ovarian cancer cells and xenograft models by inducing autophagy.

Poly (ADP-ribose) polymerase inhibitors (PARPi) can encounter resistance through various mechanisms, limiting their effectiveness. Our recent research showed that PARPi alone can induce drug resistance by promoting autophagy. Moreover, our studies have revealed that anaplastic lymphoma kinase (ALK) plays a role in regulating the survival of ovarian cancer cells undergoing autophagy. Here, we explored whether the ALK-inhibitor crizotinib could enhance the efficacy of PARPi by targeting drug-induced autophagic ovarian cancer cell and xenograft models. Our investigation demonstrates that crizotinib enhances the anti-tumor activity of PARPi across multiple ovarian cancer cells. Combination therapy with crizotinib and olaparib reduced cell viability and clonogenic growth in two-olaparib resistant cell lines. More importantly, this effect was consistently observed in patient-derived organoids. Furthermore, combined treatment with crizotinib and olaparib led to tumor regression in human ovarian xenograft models. Mechanistically, the combination resulted in increased levels of reactive oxygen species (ROS), induced DNA damage, and decreased the phosphorylation of AKT, mTOR, and ULK-1, contributing to increased olaparib-induced autophagy and apoptosis. Notably, pharmacologic, or genetic inhibition or autophagy reduced the sensitivity of ovarian cancer cell lines to olaparib and crizotinib treatment, underscoring the role of autophagy in cell death. Blocking ROS mitigated olaparib/crizotinib-induced autophagy and cell death while restoring levels of phosphorylated AKT, mTOR and ULK-1. These findings suggest that crizotinib can improve the therapeutic efficacy of olaparib by enhancing autophagy. Implications: The combination of crizotinib and PARPi presents a promising strategy, that could provide a novel approach to enhance outcomes for patients with ovarian cancer.

Knockdown of autophagy-related protein 5, ATG5, decreases oxidative stress and has an opposing effect on camptothecin-induced cytotoxicity in osteosarcoma cells.

BACKGROUND: Autophagy 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. METHODS: Autophagy-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. RESULTS: Autophagy 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. CONCLUSIONS: The 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.

Preclinical investigations of the efficacy of the glutaminase inhibitor CB-839 alone and in combinations in chronic lymphocytic leukemia.

Introduction: Chronic lymphocytic leukemia (CLL) cells are metabolically flexible and adapt to modern anticancer treatments. Bruton tyrosine kinase (BTK) and B-cell lymphoma-2 (BCL-2) inhibitors have been widely used to treat CLL, but CLL cells become resistant to these treatments over time. CB-839 is a small-molecule glutaminase-1 (GLS-1) inhibitor that impairs glutamine use, disrupts downstream energy metabolism, and impedes the elimination of reactive oxygen species. Methods: To investigate the in vitro effects of CB-839 on CLL cells, we tested CB-839 alone and in combination with ibrutinib, venetoclax, or AZD-5991 on the HG-3 and MEC-1 CLL cell lines and on primary CLL lymphocytes. Results: We found that CB-839 caused dose-dependent decreases in GLS-1 activity and glutathione synthesis. CB-839-treated cells also showed increased mitochondrial superoxide metabolism and impaired energy metabolism, which were reflected in decreases in the oxygen consumption rate and depletion of the adenosine triphosphate pool and led to the inhibition of cell proliferation. In the cell lines, CB-839 combined with venetoclax or AZD-5991, but not with ibrutinib, demonstrated synergism with an increased apoptosis rate and cell proliferation inhibition. In the primary lymphocytes, no significant effects of CB-839 alone or in combination with venetoclax, ibrutinib, or AZD-5991 were observed. Discussion: Our findings suggest that CB-839 has limited efficacy in CLL treatment and shows limited synergy in combination with widely used CLL drugs.

SIK2 inhibition enhances PARP inhibitor activity synergistically in ovarian and triple-negative breast cancers.

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.

Phosphorylated heat shock protein 27 as a potential biomarker to predict the role of chemotherapy-induced autophagy in osteosarcoma response to therapy.

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.

A Novel Compound ARN-3236 Inhibits Salt-Inducible Kinase 2 and Sensitizes Ovarian Cancer Cell Lines and Xenografts to Paclitaxel.

Purpose: Salt-inducible kinase 2 (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 the growth of 10 ovarian cancer cell lines at an IC50 of 0.8 to 2.6 µmol/L, where the IC50 of ARN-3236 was inversely correlated with endogenous SIK2 expression (Pearson 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. Clin Cancer Res; 23(8); 1945-54. ©2016 AACR.

Small molecule ErbB inhibitors decrease proliferative signaling and promote apoptosis in philadelphia chromosome-positive acute lymphoblastic leukemia.

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.