Synergistic Apoptotic Response Research Articles

Abstract 1443: The PI3K-AKT-mTOR axis persists as a therapeutic dependency in KRASG12D-driven non-small cell lung cancer

Abstract Direct inhibitors of KRASG12C and more recently KRASG12D are currently under clinical evaluation and may represent the beginning of a major translational breakthrough for non-small cell lung cancer (NSCLC) and RAS addicted cancers in general, targeting the most commonly mutated oncogene in this disease. However early reports of resistance with inactive state-selective KRASG12C inhibitors have highlighted the pressing need for rational combination partners. While preclinical combination strategies to enhance KRASG12D inhibition and combat innate resistance have already been reported for pancreatic and colorectal cancer, the application of KRASG12D inhibitors alongside combination partners in KRASG12D-driven NSCLC has not been investigated. Here we compared KRASG12C- and KRASG12D-initiated lung tumorigenesis using isogenic genetically engineered mouse models and human and mouse cell models of KRASG12C and KRASG12D-driven NSCLC initiation to compare oncopotency and signaling of each subtype. We then examined whether signaling differences observed at initiation persist in established tumors. We identified that KRASG12D is more potent than KRASG12C in inducing tumor formation. This higher potency was accompanied by co-operative activation of the PI3K-AKT-mTOR signaling axis. Disruption of this pathway using either an AKT inhibitor or mTORC1-specific inhibitor (RMC-6272, a preclinical tool compound representative of the clinical candidate RMC-5552) led to a greater reduction in transformation and proliferation in a KRASG12D−driven lung initiation model compared to the KRASG12C counterpart. Although differences between these oncogenic phenotypes become less distinguishable once tumors are established, signaling dependencies and vulnerabilities are still preserved. For instance, KRASG12C lung tumor cells are relatively more susceptible to ERK and MEK inhibition, while KRASG12D lung tumor cells are more susceptible to AKT and mTORC1 inhibition. Inhibiting the PI3K-AKT-mTOR axis alongside KRASG12D inhibition (MRTX1133) induced a robust, synergistic apoptotic response in KRASG12D tumor cell lines of varying sensitivity to KRASG12D inhibition alone, highlighting a potential combinatorial approach to override intrinsic resistance to KRASG12D inhibitors. Our preclinical data identify treatment vulnerabilities and suggest patient selection strategies for combination approaches that should be i) contextualized to individual RAS mutants, and ii) tailored to their downstream signaling programs. Citation Format: William J. McDaid, Leah Wilson, Helen Adderley, Martin Baker, Joshua Searle, Timothy Budden, Amaya Viros, Mihaela Aldea, Arianna Marinello, Jacqueline Aredo, Benjamin Besse, Heather A. Wakelee, Colin R. Lindsay, Angeliki Malliri. The PI3K-AKT-mTOR axis persists as a therapeutic dependency in KRASG12D-driven non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1443.

Genetic biomarkers predict response to dual BCL-2 and MCL-1 targeting in acute myeloid leukaemia cells.

Acute myeloid leukaemia (AML) cells often up-regulate pro-survival members of the BCL-2 protein family, such as BCL-2 and MCL-1, to avoid apoptosis. Venetoclax (ABT-199) targets BCL-2 and has shown promising efficacy in AML but over-expression of MCL-1 can cause resistance. A co-operative approach, targeting both BCL-2 and MCL-1 may therefore prove beneficial. This study investigated the potential synergistic relationship between Venetoclax and the MCL-1 inhibitor S63845 in AML cells. We treated MV4-11 cells and primary AML samples for 4 hours with Venetoclax, S63845 or the combination. We used a short-term flow cytometric technique to assess synergy using cytochrome C release as a read out of response. The combination of Venetoclax and S63845 produced a synergistic apoptotic response in MV4-11 cells and primary samples, including the leukaemia re-populating leukaemic stem cell (LSC) population, in 92% of the samples. Known molecular biomarkers of response to BCL-2 and MCL-1 targeting agents were corroborated, and augmented, with the short-term functional assay. The assay also predicted potential biomarkers of response to the combination of BCL-2 and MCL-1 targeting agents. Primary samples with an IDH2_140 mutation were more sensitive to Venetoclax as a single agent whereas samples with a FLT3-ITD mutation were more resistant. This resistance could be reversed when combined with S63845. All FLT3-ITD and NPM1 mutated samples were sensitive to the combination of drugs.We report that co-operatively targeting BCL-2 and MCL-1 may be beneficial in AML and a short-term in vitro assay can identify patients who might best respond to this combination.

The Combination of Metformin and Valproic Acid Induces Synergistic Apoptosis in the Presence of p53 and Androgen Signaling in Prostate Cancer.

We investigated the potential of combining the hypoglycemic drug metformin (MET) and the antiepileptic drug valproic acid (VPA), which act via different biochemical pathways, to provide enhanced antitumor responses in prostate cancer. Prostate cancer cell lines (LNCaP and PC-3), normal prostate epithelial cells (PrEC), and patient-derived prostate tumor explants were treated with MET and/or VPA. Proliferation and apoptosis were assessed. The role of p53 in response to MET + VPA was assessed in cell lines using RNAi in LNCaP (p53+) and ectopic expression of p53 in PC-3 (p53-). The role of the androgen receptor (AR) was investigated using the AR antagonist enzalutamide. The combination of MET and VPA synergistically inhibited proliferation in LNCaP and PC-3, with no significant effect in PrEC. LNCaP, but not PC-3, demonstrated synergistic intrinsic apoptosis in response to MET + VPA. Knockdown of p53 in LNCaP (p53+, AR+) reduced the synergistic apoptotic response as did inhibition of AR. Ectopic expression of p53 in PC-3 (p53-, AR-) increased apoptosis in response to MET + VPA. In patient-derived prostate tumor explants, MET + VPA also induced a significant decrease in proliferation and an increase in apoptosis in tumor cells. In conclusion, we demonstrate that MET + VPA can synergistically kill more prostate cancer cells than either drug alone. The response is dependent on the presence of p53 and AR signaling, which have critical roles in prostate carcinogenesis. Further in vivo/ex vivo preclinical studies are required to determine the relative efficacy of MET + VPA as a potential treatment for prostate cancer. Mol Cancer Ther; 16(12); 2689-700. ©2017 AACR.

Abstract LB-233: Combination of metformin and valproic acid in personalized prostate cancer treatment: the role of p53 and androgen receptor signaling

Abstract The anti-diabetic drug Metformin (MET) has been shown to reduce the incidence of prostate cancer, while the anti-epileptic drug Valproic Acid (VPA) has also been shown to have an anti-tumor effect. Here we aimed to determine if the combination of MET and VPA would induce at least an additive anti-tumour effect in prostate cancer due to their different mechanisms of action, and whether common mutations in prostate cancer (e.g. TP53 and AR genes) would affect the response. In in vitro experiments, the proliferation and apoptosis responses of LNCaP (p53+, AR+), PC-3 (p53null, ARmut), DUCaP (p53mut, AR+) and Prostate Epithelial Cell (PrEC) lines to MET and/or VPA was assessed using an IncuCyte® FLR system. The Drug Combination Index (CI) was calculated using the Chou-Talalay method. mRNA knock-down of TP53 in LNCaP was performed using siRNA. p53 plasmid was transfected into PC3 using Lipofectamine 3000 and cell death was investigated using nuclear fragmentation index and cleaved-caspase-3 intensity in an Operetta® High Content Imaging System. AR was chemically inhibited using Enzalutamide. In a patient derived prostatic explant model, cell proliferation and apoptosis were determined by Ki67 and cleaved-caspase 3 immunohistochemistry. The combination of MET+VPA synergistically inhibited proliferation in LNCaP (CI = 0.5) and PC-3 (CI = 0.6), with only an additive inhibition in DUCaP. The combination of MET and VPA also induced a significant decrease in Ki67 and increase in cleaved-caspase 3 compared to vehicle control in the human prostatic explant model (n = 7). Only LNCaP demonstrated a significantly higher (12-fold) apoptosis response to MET+VPA including at concentrations lower than the human toxic threshold (0.5 mM MET + 2.5 mM VPA), whereas the difference was not significant in PC3, DUCaP and PrEC cells. Knock-down of p53 in LNCaP reduced the synergistic apoptotic response, while maintaining the same anti-proliferative effect. p53 plasmid transfection into PC3 induced increased cell death in response to Met+VPA. AR inhibition in LNCaP reduced the synergistic apoptosis response and increased the anti-proliferative effect. AR inhibition had no effect in PC-3, an androgen-resistant cell line. The concentration of MET and VPA used here are below the human plasma toxic threshold and may be a useful treatment for prostate cancer, particularly in tumours with functional p53 and/or AR signalling. As both drugs are currently used for non-cancer indications, repurposing of these drugs could be rapidly integrated into clinical use. Future functional studies include investigation of phosphorylation and acetylation changes of the p53 protein, and confirmation of the results in xenograft mice and a phase I clinical trial. Citation Format: Linh N.k Tran, Ganessan Kichenadasse, Katherine L. Morel, Rebecca J. Ormsby, Lisa M. Butler, Maggie M. Centenera, Pamela J. Sykes. Combination of metformin and valproic acid in personalized prostate cancer treatment: the role of p53 and androgen receptor signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-233.

Sequential co-delivery of miR-21 inhibitor followed by burst release doxorubicin using NIR-responsive hollow gold nanoparticle to enhance anticancer efficacy

Previous literature and our study showed the delivery sequence of microRNA inhibitor and chemotherapeutic compounds achieve distinct therapeutic anticancer efficacy. Yet, it is challenging to use nanoparticle to achieve sequential drug delivery. In the current study, we designed sequential co-delivery system using a near-infrared-radiation (NIR) responsive hollow gold nanoparticle (HGNPs) to achieve sequential release of microRNA inhibitor (miR-21i)/doxirubicin(Dox) in order to achieve synergistic efficacy. PAMAM modified HGNPs was used to encapsulate miR-21i and Dox. Upon entering tumor cells, miRNA-21i was released first to sensitize the cancer cells, the subsequent burst release of Dox was achieved by NIR triggered collapse of HGNPs. This sequential delivery of miRNA-21i and Dox produced a synergistic apoptotic response, thereby enhancing anticancer efficacy by 8-fold and increasing anti-cancer stem cell activity by 50-fold. The sequential delivery of miR-21i and Dox using HGNPs under NIR after intravenous administration showed high tumor accumulation and significantly improved efficacy, which was 4-fold compared to free Dox group. These data suggested that the sequential co-delivery of miR-21i followed by burst release Dox using NIR-responsive HGNPs sensitized cancer cells to chemotherapeutic compound, which provided a novel concept for co-delivery miRNA inhibitors and chemotherapeutic compounds to enhance their efficacy.

The Mutant KRAS Gene Up-regulates BCL-XL Protein via STAT3 to Confer Apoptosis Resistance That Is Reversed by BIM Protein Induction and BCL-XL Antagonism

In colorectal cancers with oncogenic GTPase Kras (KRAS) mutations, inhibition of downstream MEK/ERK signaling has shown limited efficacy, in part because of failure to induce a robust apoptotic response. We studied the mechanism of apoptosis resistance in mutant KRAS cells and sought to enhance the efficacy of a KRAS-specific MEK/ERK inhibitor, GDC-0623. GDC-0623 was shown to potently up-regulate BIM expression to a greater extent versus other MEK inhibitors in isogenic KRAS HCT116 and mutant KRAS SW620 colon cancer cells. ERK silencing enhanced BIM up-regulation by GDC-0623 that was due to its loss of phosphorylation at Ser(69), confirmed by a BIM-EL phosphorylation-defective mutant (S69G) that increased protein stability and blocked BIM induction. Despite BIM and BIK induction, the isogenic KRAS mutant versus wild-type cells remained resistant to GDC-0623-induced apoptosis, in part because of up-regulation of BCL-XL. KRAS knockdown by a doxycycline-inducible shRNA attenuated BCL-XL expression. BCL-XL knockdown sensitized KRAS mutant cells to GDC-0623-mediated apoptosis, as did the BH3 mimetic ABT-263. GDC-0623 plus ABT-263 induced a synergistic apoptosis by a mechanism that includes release of BIM from its sequestration by BCL-XL. Furthermore, mutant KRAS activated p-STAT3 (Tyr(705)) in the absence of IL-6 secretion, and STAT3 knockdown reduced BCL-XL mRNA and protein expression. These data suggest that BCL-XL up-regulation by STAT3 contributes to mutant KRAS-mediated apoptosis resistance. Such resistance can be overcome by potent BIM induction and concurrent BCL-XL antagonism to enable a synergistic apoptotic response.

Role of X-Linked Inhibitor of Apoptosis as a Prognostic Marker and Therapeutic Target in Papillary Thyroid Carcinoma.

Papillary thyroid cancer (PTC) is the second most common cancer in females in Saudi Arabia. However, the pathogenesis of PTC is still not fully elucidated. To identify potential genes that play important role in progression of PTC, we studied the role of X-linked inhibitor of apoptosis protein (XIAP) as a potential prognostic marker and therapeutic target in a large cohort of PTC samples and cell lines. A DNA microarray chip was used to screen for gene copy number. XIAP expression was assessed by immunohistochemistry in a tissue microarray format on a cohort of 1022 clinical samples. In vitro and in vivo studies were performed using Embelin and/or LY294002 on PTC cell lines. XIAP was found to be amplified in 14 of 29 and overexpressed in 48.8% of PTC cases. XIAP overexpression was significantly associated with old age, extrathyroidal extension, tumor size, nodal involvement, tall-cell variant, advanced stage disease, and significantly poor disease-free survival (P = .0341). XIAP was also significantly associated with phosphorylated AKT (P < .0001), Bcl-Xl (P < .0001), and Ki67 (P = .0006) proteins. Embelin treatment caused growth inhibition and apoptosis in PTC cell lines and induced tumor regression in PTC xenograft in nude mice. Finally, the combination of suboptimal doses of Embelin and LY294002 induced a synergistic apoptotic response in PTC cells. XIAP dysregulation in PTC confers an aggressive phenotype with poor outcome. In vitro and in vivo studies using an XIAP inhibitor suggest that this subgroup of PTC with overexpression of XIAP can be therapeutically targeted, either alone or in combination, to induce efficient apoptosis in these cancers.

Molecular target therapy of AKT and NF-kB signaling pathways and multidrug resistance by specific cell penetrating inhibitor peptides in HL-60 cells.

PI3/AKT and NF-kB signaling pathways are constitutively active in acute myeloid leukemia and cross-talk between the two has been shown in various cancers. However, their role in acute myeloid leukemia has not been completely explored. We therefore used cell penetrating inhibitor peptides to define the contributions of AKT and NF-kB to survival and multi drug resistance (MDR) in HL-60 cells. Inhibition of AKT and NF-kB activity by AKT inhibitor peptide and NBD inhibitor peptide, respectively, resulted in decreased expression of mRNA for the MDR1 gene as assessed by real time PCR. In addition, treatment of HL-60 cells with AKT and NBD inhibitor peptides led to inhibition of cell viability and induction of apoptosis in a dose dependent manner as detected by flow cytometer. Finally, co-treatment of HL-60 cells with sub-optimal doses of AKT and NBD inhibitor peptides led to synergistic apoptotic responses in AML cells. These data support a strong biological link between NF-kB and PI3-kinase/AKT pathways in the modulation of anti- apoptotic and multi drug resistant effects in AML cells. Synergistic targeting of these pathways using NF-kB and PI3-kinase/AK inhibitor peptides may have a therapeutic potential for AML and possibly other malignancies with constitutive activation of these pathways.

Abstract P6-04-17: The irreversible c-Jun N-terminal kinase (JNK) inhibitor, JNK-IN-8, sensitizes basal-like breast cancer cells to lapatinib

Abstract Basal-like breast cancers represent 15-20% of breast cancers diagnosed each year. These tumors appear earlier in life and have the worst prognosis due to high prevalence of metastasis. Endocrine and molecularly targeted therapies such as trastuzumab or lapatinib are ineffective against this tumor subtype. A high frequency of p53 mutations, low BRCA1 expression along with high epidermal growth factor receptor (EGFR) expression provide some insight into what types of targeted therapies may be useful for basal-like tumors in the future. We have recently shown that JNK2 mediates the expression of p53 and BRCA1 in response to EGFR activation to promote epithelial to mesenchymal transition and metastasis. Further, high JNK2 expression is associated with poorer survival in patients with basal-like breast cancer. These findings suggest that the JNK pathway may be a promising target for basal-type breast cancer treatment. Commonly used competitive ATP inhibitors of JNK have suffered from lack of specificity for JNK. However, the Gray laboratory recently developed highly specific covalent inhibitors of JNK (Zhang, et al. 2012). The agent showing the best properties is JNK-IN-8. Our data show that treatment of human and mouse basal-like, breast cancer cell lines with JNK-IN-8 sensitizes them to lapatinib. Single agents had minor affects on cell viability and cell cycle regulation, but combination treatment led to G2/M arrest and endoreduplication along with synergistic apoptotic responses. Further, the expression and electrophoretic mobility of mitotic arrest deficient 2 (MAD2) (a protein mediating chromosomal segregation) decreased after combination treatment which could explain the chromosomal duplication abnormalities observed. Combination treatment using lapatinib and JNK-IN-8 modulates extracellular signal-regulated kinase (ERK) phosphorylation unlike either drug alone suggesting that both JNK and ERK target G2/M check point proteins, and when perturbed, sensitize basal-type breast cancer cells to targeted therapies. We conclude that use of specific JNK inhibitors in tumors that are resistant to lapatinib, or perhaps trastuzumab, may sensitize them to treatments with these drugs, and may also prevent resistance to these drugs in tumors that were initially responsive. Considering that JNKs are nodes in receptor tyrosine kinase and many other signaling pathways, JNK-IN-8 treatment may improve the efficacy of other targeted therapies as well. Zhang, T. et al. “Discovery of potent and selective covalent inhibitors of JNK”. Chem Biol. 2012 Jan 27;19(1):140-54. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-17.

Synergistic apoptotic response between valproic acid and fludarabine in chronic lymphocytic leukaemia (CLL) cells involves the lysosomal protease cathepsin B

Fludarabine, a nucleoside analogue, is commonly used in combination with other agents for the treatment of chronic lymphocytic leukaemia (CLL). In previous studies, valproic acid (VPA), an inhibitor of histone deacetylases, combined with fludarabine to synergistically increase apoptotic cell death in CLL cells. In the present study, we found that the combination of fludarabine and VPA decreases the level of the anti-apoptotic proteins Mcl-1 and XIAP in primary CLL cells. Treatment with fludarabine alone, or in combination with VPA, led to the loss of lysosome integrity, and chemical inhibition of the lysosomal protease cathepsin B, using CA074-Me, was sufficient to reduce apoptosis. VPA treatment increased cathepsin B levels and activities in primary CLL cells, thereby priming CLL cells for lysosome-mediated cell death. Six previously treated patients with relapsed CLL were treated with VPA, followed by VPA/fludarabine combination. The combined therapy resulted in reduced lymphocyte count in five out of six and reduced lymph node sizes in four out of six patients. In vivo VPA treatment increased histone-3 acetylation and cathepsin B expression levels. Thus, the synergistic apoptotic response with VPA and fludarabine in CLL is mediated by cathepsin B activation leading to a decrease in the anti-apoptotic proteins.

Abstract 597: The combination of valproic acid and fludarabine treatment induces a synergistic apoptotic response in chronic lymphocytic leukemia (CLL) patients involving the lysosomal protease cathepsin B.

Abstract Chronic Lymphocytic Leukaemia (CLL) is the most common hematological malignancy in the western world. Fludarabine, a nucleoside analogue, is commonly used in combinational treatments for CLL. However, patients commonly develop resistance to fludarabine based therapies. Valproic Acid (VPA), an inhibitor of histone deacetylases (HDACs), showed synergistic apoptotic responses when combined with fludarabine in human leukemic cells and primary CLL cells. The mechanism for this synergistic apoptotic response is unknown. We found that fludarabine and VPA treatment increased activates caspases-8, -9, -3, and caspase-2 and a decreased in expression of anti-apoptotic proteins Mcl-1 and XIAP. Treatment with fludarabine alone or in combination with VPA led to the loss of lysosome integrity suggesting a leakage of the lysosomal content into the cytosol in response to these drugs. Chemical inhibition of a specific lysosomal protease, cathepsin B, using CA074-Me was sufficient to stabilize Mcl-1 and XIAP while reducing caspase activation and apoptosis. Addition of purified cathepsin B to leukemic cell lysates led to the reduction in protein levels of Mcl-1, XIAP and pro-caspase-2, thus suggesting that the re-localization of cathepsin B into the cytosol is sufficient to degrade these proteins. VPA treatment increased cathepsin B levels in both leukemic cell lines and primary CLL cells. VPA also increased cathepsin B activity. In addition, six relapsed CLL patients who had received at least one prior therapy with fludarabine were treated with VPA alone or in combination with fludarabine. No responses were seen after 28 days using VPA alone but in five patients who continued on VPA with fludarabine, three patients showed reduced lymphocyte count after receiving at most five cycles of the combination therapy. When the CLL cells from VPA-treated CLL patients were examined, VPA administration induced increased levels of histone-3 acetylation and cathepsin B expression in vivo. Thus, VPA and fludarabine synergistic apoptotic response is mediated by cathepsin B activation leading to a decrease in anti-apoptotic proteins in CLL cells and likely contributes to the clinical response observed in relapsed, fludarabine-refractory CLL patients. Citation Format: Ju Yoon Yoon, David Szwajcer, Ganchimeg Ishdorj, Pat Benjaminson, Rajat Kumar, James B. Johnston, Spencer B. Gibson. The combination of valproic acid and fludarabine treatment induces a synergistic apoptotic response in chronic lymphocytic leukemia (CLL) patients involving the lysosomal protease cathepsin B. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 597. doi:10.1158/1538-7445.AM2013-597

Abstract 3388: Novel aurora plus Bruton's tyrosine kinase-targeted therapies for aggressive B-cell non-Hodgkin's lymphoma.

Abstract Exploring synthetic lethality in the context of signaling pathways with novel targeted agents may yield effective combination therapies for aggressive B-NHL. Auroras are a family of mitotic oncogenic serine/threonine kinases intimately involved in high fidelity regulation of cell division. We previously demonstrated that aberrant Aurora (A and B) expression portends a poor survival in mantle cell lymphoma (MCL) patients. Aberrant aurora expression leads to genetic instability, polyploidy, and resistance to microtubule targeted agents (MTAs). We demonstrated that combination of an Aurora inhibitor MLN8237 (alisertib) or AT9283 with MTAs (e.g. taxol or vincas) leads to a mechanistic decoupling of the mitotic spindle manifesting as a synergistic apoptotic response in cell culture and in mouse xenograft models of aggressive B-NHL. Gene expression profiling of harvested mouse xenograft tumors at the end of treatment (3 weeks) with an aurora inhibitor + MTA showed over-expression of several markers of resistance of which BTK (Bruton's tyrosine kinase) was the most prominent. BTK plays a role in uncontrolled activation and proliferation of malignant B-cells via the chronic active B-cell receptor pathway in aggressive B-NHL. We hypothesized that targeting proliferation [BTK] and replication [Aurora] would be a novel therapeutic strategy in aggressive B-NHL. We demonstrate that both aurora (A and B) and BTK are highly over-expressed in B-NHL cell lines (Granta-519, SUDHL4, SUDHL10, OCI-Ly-10, TMD8, U-2932) and harvested mouse xenograft tumour treated with an Aurora inhibitor + vincristine and in patient samples. IC50 values for AT9283 is 5-10nM, and CNX652 is 0.07-5μM. The combination index (CI) indicated significant synergism for AT9283 plus CNX-652 or vice versa. Combination therapy showed increased apoptosis (flow cytometry, PARP-cleavage) in a dose-dependent manner. Both AT9283 and AVL-292 (CNX-652 is a structural analog with similar in vitro and in vivo properties to AVL-292) are in early phase clinical trials in hematologic malignancies. We utilized clinically relevant doses to evaluate the efficacy of single agent and combination therapy of AT9283 and CNX652 in mouse xenograft models (Granta-519) of MCL. The first mouse model evaluated single agent dose-response and target modulation by Western blotting (pHisH3, pBTK) in tumors harvested at the end of 3 weeks of treatment (CNX-652 daily oral for 3 weeks or AT9283 daily IV for 3 weeks, n=12 mice per cohort). The second study evaluated the combination of AT9283 and CNX-652 for endpoints of response, overall survival and target modulation. Gene expression and protein analysis of harvested tumors at the end of treatment (3 weeks) have been interrogated to ascertain the mechanistic role of Aurora plus BTK inhibition. The combination of AT9283 + CNX652 represents a novel therapeutic strategy for aggressive B-NHL [Funded by the Hope Foundation]. Citation Format: Daruka Mahadevan, Carla Morales, Russell Karp, Laurence Cooke, John Lyons, Juswinder Singh, Wenqing Qi. Novel aurora plus Bruton's tyrosine kinase-targeted therapies for aggressive B-cell non-Hodgkin's lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3388. doi:10.1158/1538-7445.AM2013-3388

Abstract 526: Combined inhibition of mTOR and AKT activity induces potent apoptosis in epithelial ovarian cancer cells .

Abstract Mammalian target of rapamycin (mTOR) and Phosphatidylinositol 3-kinase (PI3K) are two key components of the PI3K/Akt/mTOR signaling pathway. These two signal transduction cascades regulate a number of physiological cell functions including differentiation, proliferation, apoptosis and autophagy. Dysregulation of these pathways have been found in many cancers including DLBCL, thyroid cancer, breast cancer, prostate cancer and lung cancer. mTOR has been shown to activate the PI3-kinase/AKT survival pathway and understanding the interplay among signaling molecules in the PI3K/Akt/mTOR pathway is of utmost importance. Two distinct mTOR complexes; mTORC1 and mTORC2, have been identified. mTORC1 is downstream of Akt, sensitive to rapamycin inhibition, and controls cap-dependent protein translation. In contrast, mTORC2 is directly upstream of Akt and is resistant to rapamycin. In this study, we investigated the effect of mTOR inhibition on cell viability and induction of apoptosis in epithelial ovarian cancer (EOC) cells, either alone or in combination with PI3-kinase/AKT inhibitor. We found that Torin2, an mTOR inhibitor, decreased cell viability and induced apoptosis in EOC cell lines in a dose dependent manner. Torin2 also inactivated mTOR1 and mTOR2 as well as downstream targets of mTOR; 4E-BP1 and p70S6. Torin2 treatment of EOC cell lines also induced mitochondrial dependent apoptosis via activation and cleavage of caspases. Finally, combination treatment of EOC cells with Torin2 and LY294002, a PI3-kinase/AKT inhibitor induced a synergistic apoptotic response via inactivation of AKT, mTOR1 and mTOR2 and activation of caspases. These studies will give us an insight on the molecular relationship between these survival pathways and suggest that simultaneous inhibition of mTOR and PI3-kinase/AKT pathways may be beneficial for the management of EOC. Citation Format: Azhar R. Hussain, Shahab Uddin, Maqbool Ahmed, Khawla S. Al-Kuraya. Combined inhibition of mTOR and AKT activity induces potent apoptosis in epithelial ovarian cancer cells . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 526. doi:10.1158/1538-7445.AM2013-526

Cross-Talk between NFkB and the PI3-Kinase/AKT Pathway Can Be Targeted in Primary Effusion Lymphoma (PEL) Cell Lines for Efficient Apoptosis

BackgroundA number of constitutively activated signaling pathways play critical roles in the survival and growth of primary effusion lymphoma cells (PELs) including NFkB and PI3/AKT kinase cascades. NFkBis constitutively activated in a number of malignancies, including multiple myeloma, Burkitt’s lymphoma and diffuse large cell B-cell lymphoma. However, its role in primary effusion lymphoma has not been fully explored.Methodology/Principal FindingsWe used pharmacological inhibition and gene silencing to define the role of NFkB in growth and survival of PEL cells. Inhibition of NFkB activity by Bay11-7085 resulted in decreased expression of p65 in the nuclear compartment as detected by EMSA assays. In addition, Bay11-7085 treatment caused de-phosphorylation of AKT and its downstream targets suggesting a cross-talk between NFkB and the PI3-kinase/AKT pathway. Importantly, treatment of PEL cells with Bay11-7085 led to inhibition of cell viability and induced apoptosis in a dose dependent manner. Similar apoptotic effects were found when p65 was knocked down using specific small interference RNA. Finally, co-treatment of PEL cells with suboptimal doses of Bay11-7085 and LY294002 led to synergistic apoptotic responses in PEL cells.Conclusion/SignificanceThese data support a strong biological-link between NFkB and the PI3-kinase/AKT pathway in the modulation of anti-apoptotic effects in PEL cells. Synergistic targeting of these pathways using NFKB- and PI3-kinase/AKT- inhibitors may have a therapeutic potential for the treatment of PEL and possibly other malignancies with constitutive activation of these pathways.

γ-Secretase inhibition promotes cell death, Noxa upregulation, and sensitization to BH3 mimetic ABT-737 in human breast cancer cells

IntroductionInappropriate Notch signaling, downstream of γ-secretase activity, is understood to have tumor-promoting function and to be associated with poor outcome in cancer, of the breast in particular. The molecular basis of antitumoral effects of its inhibitors, however, remains poorly characterized. Moreover, the effects of their combination with the pro-apoptotic pharmacologic inhibitor of Bcl-2/Bcl-xL, ABT-737, have never been evaluated. In this study, we thus specifically addressed the biologic consequences of targeting γ-secretase and Bcl-2/Bcl-xL, alone or simultaneously, in breast cancer cell lines as well as in a novel human breast cancer ex vivo assay.MethodsBy using in vitro 2D or 3D cultures of breast cancer cells plus a novel preclinical short-term ex vivo assay that correctly maintains human mammary tissue integrity and preserves tumor microenvironment, we tested the effects of the pharmacologic γ-secretase inhibitor GSIXII used as a single agent or in combination with ABT-737.ResultsWe show herein that the γ-secretase inhibitor, GSIXII, efficiently induces apoptosis in breast cancer cell lines by a process that relies on the induction of Noxa, a pro-apoptotic Bcl2-homology 3 domain (BH3)-only protein of the Bcl-2 family that functions as an inhibitor of antiapoptotic Mcl1. GSIXII also targets mammary cancer stem-like cells because it dramatically prevents in vitro mammosphere formation. Moreover, combining GSIXII treatment with ABT-737, a BH3-mimetic inhibitor of additional antiapoptotic proteins, such as Bcl-2 and Bcl-xL, leads to both a synergistic apoptotic response in breast cancer cells and to an inhibitory effect on mammosphere formation. These effects are also found when a Notch transcriptional inhibitor, SAHM1, is used. Finally, we evaluated individual human tumor responses to γ-secretase inhibition alone or in combination with ABT-737 in ex vivo assays. Analysis of a series of 30 consecutive tumors indicated that a majority of tumors are sensitive to apoptosis induction by GSIXII and that association of GSIXII with ABT-737 leads to an enhanced induction of apoptosis in tumor cells.ConclusionsWe thus provide evidence that γ-secretase, and downstream Notch signaling, are relevant targets in breast cancer. GSIXII, used as single agent or in combination with clinically relevant BH3-mimetics, is a promising innovative proapoptotic strategy to treat mammary tumors.

Death receptor 4 is preferentially recruited to lipid rafts in chronic lymphocytic leukemia cells contributing to tumor necrosis related apoptosis inducing ligand-induced synergistic apoptotic responses

Tumor necrosis related apoptosis inducing ligand receptor 1 (TRAIL-R1, death receptor 4 [DR4]) and TRAIL-R2 (DR5) have been proposed as targets for cancer therapy, but which death receptor to target for chemotherapy in chronic lymphocytic leukemia (CLL) is uncertain. Herein, we discovered that Burkitt lymphoma B cell line, BJAB, CLL-like cell line, I-83, and pre-acute lymphocytic leukemia B cell line, NALM-6, underwent apoptosis following TRAIL, whereas a CLL-like cell line, JMV-3, and primary CLL cells failed to undergo apoptosis. In TRAIL resistant CLL cells, only activation of DR4 provided an increase in fludarabine induced apoptosis. This was mediated in part by the localization of DR4 but not DR5 in lipid rafts following TRAIL and fludarabine treatment. This preference for DR4 activation leading to increased fludarabine induced apoptosis was also observed following SAHA, PS-341, and chlorambucil treatment in primary CLL cells. Thus, CLL cells selectively activate DR4 partially mediated through its localization to lipid rafts leading to apoptosis when combined with chemotherapeutic drugs.

Synergistic killing of colorectal cancer cells by oxaliplatin and ABT-737

Oxaliplatin is frequently used in the treatment of metastatic colorectal cancer (CRC). Our previous work shows that oxaliplatin induces the pro-apoptotic protein Noxa in CRC cells. The Bcl2-inhibitor ABT-737 is particularly effective in cells with high Noxa levels. Therefore, we tested whether oxaliplatin and ABT-737 display synergy in killing CRC cells. A panel of CRC cell lines was treated with oxaliplatin and ABT-737, either alone or in combination. Apoptosis was measured by FACS analysis of sub-G1 DNA content and by Western blot analysis of caspase-3 processing. Noxa expression was suppressed by lentiviral RNA interference. Oxaliplatin and ABT-737 displayed a strong synergistic apoptotic response, which was dependent on wildtype TP53 and oncogenic KRAS. TP53 and KRAS were required for drug-induced Noxa expression and this was essential for tumor cell apoptosis. Oxaliplatin, but not ABT-737, induced p53 accumulation, but both drugs stimulated Noxa expression. Combination treatment of mice with subcutaneous tumor xenografts drastically reduced tumor volume, while single drug treatment had no effect. ABT-737 synergizes with oxaliplatin to kill colorectal cancer cells. This requires induction of Noxa by wildtype TP53 and oncogenic KRAS. Future studies should explore the anti-tumor efficacy of this drug combination in mouse models for spontaneous CRC development and in patient-derived tumor cell cultures and xenografts.

Interactions of the Mdm2/p53 and Proteasome Pathways: Implications for Combination Strategies to Enhance the Anti-Myeloma Activity of Bortezomib

The “guardian of the genome” p53 is inactivated in most human cancers via missense mutations in its DNA binding core domain or via overexpression of the human homolog of Mdm2 (Hdm2), an E3 ubiquitin ligase that binds and ubiquitinates p53 thereby leading to its degradation via the ubiquitin/proteasome pathway. Direct inhibition of Mdm2 function could potentially stabilize p53 and activate the p53 apoptotic pathway, which could be a useful non-genotoxic approach for the treatment of cancer. Nutlin-3, a cis-imidazoline small molecule with affinity for the p53-binding pocket of Mdm2 and capable of disrupting the p53-Mdm2 interaction, can activate p53 and induce apoptosis in vitro in many malignancies, including multiple myeloma (MM) cells. We hypothesized that suppression of Mdm2-mediated p53 ubiquitination may synergize with accumulation of p53 triggered by bortezomib. To address this question, we evaluated the response of MM cells vs. select models of epithelial cancers to bortezomib and its combination with nutlin-3. We observed that breast (MDA-MB-231-luc); prostate (DU145); thyroid (SW579, FRO, WRO, TT) and colon (ARO/HT-29) cancer cell lines exhibited synergistic apoptotic response to the combination of sublethal concentrations of bortezomib plus nutlin-3. This synergistic killing effect was associated with synergistic increase in the expression of p53, p21, Mdm2, Bax, Noxa, PUMA and the cleavage of caspase-3, caspase-9 and PARP. In contrast, MM cell lines (MM-1S, MM-1R, NCI-H929, KMS-11) exhibited mostly an additive effect when treated with the combination of bortezomib plus nutlin-3. To further probe the biological relevance of these findings in the context of the bone microenvironment in MM, we compared the transcriptional profile of p53 target genes as well as the response to nutlin-3 in MM cells cultured alone vs. in the presence of bone marrow stromal cells (BMSCs). We observed that co-culture with BMSCs attenuates the response of MM cells to single-agent nutlin-3 and is associated, in p53 wild-type cells, with increased amplitude of the transcriptional signature of genes suppressed by activated p53, suggesting that BMSCs suppress p53 activity in MM cells. Overall, these results suggest a complex correlation between the Mdm2/p53 and proteasome pathways: many p53 mutant epithelial cancer models can become more bortezomib-responsive by Mdm2 inhibition, while MM cells, with higher baseline responsiveness to bortezomib and nutlin-3, typically do not exhibit significant increases in their response to the combination of these 2 drug classes compared to the single-agent treatments. These observations suggest that concurrent Mdm2 inhibition may extend the spectrum of bortezomib applications to tumor types with currently limited single-agent response to proteasome inhibition. In MM, p53 mutations have been historically considered to be present in only late stage disease (e.g. plasma cell leukemia/extramedullary MM), but their prevalence may increase in the future as more patients with advanced MM survive longer thanks to recently introduced drug classes (thalidomide, bortezomib, lenalidomide). In those MM patients who harbor p53 pathway lesions and have developed bortezomib-resistance/refractoriness, combinations of bortezomib with Mdm2 inhibitors may also represent in the future an intriguing potential therapeutic option that merits further preclinical and clinical evaluation.

Lysophosphatidic Acid Protects Cancer Cells from Histone Deacetylase (HDAC) Inhibitor-induced Apoptosis through Activation of HDAC

Histone deacetylases (HDACs) catalyze the removal of acetyl groups from histones and contribute to transcriptional repression. In addition, the HDAC inhibitors induce apoptosis in cancer cells through alterations in histone acetylation and activation of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) apoptotic pathway. Lysophosphatidic acid (LPA) is a growth factor that promotes survival of cancer cells through activation of G protein-coupled receptors. Here we show that HDAC inhibitors can induce apoptosis through activation of the TRAIL apoptotic pathway, and LPA prevented HDAC inhibitor-induced apoptosis and increased TRAIL receptor DR4 (death receptor 4) protein expression. This was associated with increased HDAC1 recruitment to the DR4 promoter following LPA treatment and a reduction in HDAC inhibitor-induced histone acetylation in the DR4 promoter. In addition, LPA induces HDAC enzyme activity in a dose- and time-dependent manner, and this is associated with HDAC1 activation and increased binding of HDAC1 to HDAC2. Reducing the expression of HDAC1 significantly lowered LPA-induced HDAC activity and increased histone acetylation. LPA induction of HDAC activity was blocked by the LPA receptor antagonist, Ki16425, or by inhibiting receptor activation with pertussis toxin. Reducing the expression of the LPA receptor LPA(1) also blocked LPA-induced HDAC activation. In addition, LPA reduced histone acetyltransferase enzymatic activity. Finally, LPA attenuated the ability of the HDAC inhibitor to reduce HDAC activity. Thus, LPA enhances survival of cancer cells by increasing HDAC activity and reducing histone acetylation.

Death receptor-4 (DR4) expression is regulated by transcription factor NF-κB in response to etoposide treatment

Tumour necrosis factor related apoptosis inducing ligand (TRAIL) binds to death receptor 4 (DR4) activating the apoptotic signalling pathway. DNA damaging agents (genotoxins) such as etoposide increase DR4 expression and when combined with TRAIL induce a synergistic apoptotic response. The mechanism for up-regulation of DR4 expression following genotoxin treatment is not well understood. Herein, we determined that transcription factor NF-kappaB plays a role in genotoxin induced DR4 expression. Increased expression of DR4 following etoposide treatment is blocked by inhibition of the NF-kappaB pathway. Moreover, expression of the p65 subunit of NF-kappaB is sufficient to increase DR4 protein levels. Indeed, knockdown of p65 by RNA interference blocked etoposide up-regulation of DR4. We further identified a functional NF-kappaB binding site located in the DR4 promoter. Mutation of this site abrogates the induction of luciferase activity after p65 over-expression. Furthermore, electromobility shift assays and chromatin immunoprecipitaton suggest that NF-kappaB binds to this site upon etoposide treatment. MEK kinase 1 (MEKK1) is a serine threonine kinase that is activated following etoposide treatment and activates NF-kappaB. Expression of the kinase inactive MEKK1 (MEKK1-KM) abrogates the up-regulation of DR4 after etoposide treatment. Taken together, NF-kappaB plays a role in up-regulation of DR4 following etoposide treatment.